Alpha-1-microglobulin derived peptide fragments and uses thereof

Abstract

The present disclosure relates to agents comprising polypeptides and their use in medicine. Specifically, it relates to fragments of alpha-1-microglobulin. Disclosed herein are also fusion proteins comprising said polypeptide, polynucleotides encoding said polypeptide, vectors comprising said polynucleotide, and cells comprising said polynucleotides or said vectors.

Description

[0001] ALPHA-1 -MICROGLOBULIN DERIVED PEPTIDE FRAGMENTS AND USES

[0002] THEREOF

[0003] Technical field

[0004] The present disclosure relates to agents comprising a polypeptide and their use in medicine. Specifically, it relates to polypeptide fragments of alpha-1 -microglobulin or variants thereof.

[0005] Background

[0006] Alpha-1 -microglobulin (a1 -microglobulin, A1 M, protein HC) is a protein with an extracellular tissue-cleaning function (Ekstrom etal., 1977; Akerstrom and Gram, 2014). It is present in many animals, such as fish, birds, rodents, mammals and other vertebrates. A1 M is synthesized mainly in the liver but also at a lower rate in most other cells in the body. It is encoded by the ai-microglobulin-bikunin precursor gene (AMBP) and translated in all cells and species as a continuous peptide precursor together with another protein, bikunin (Kaumeyer et al., 1986).

[0007] A1 M is a physiological antioxidant, active both inside cells and in the extracellular compartments. Four different molecular mechanisms have been described to form the basis for protective effects of A1 M: 1 ) A catalytic reductase activity, 2) a radical-trapping mechanism, 3) heme binding and degradation, and 4) binding to and protection of mitochondria. Utilizing its redox properties, A1 M operates as a “circulating wastebasket” capable of removing reactive oxygen species (ROS) and other pathological oxidation products from the body by binding and transporting them to the kidneys, where they are degraded. In particular, the heme-scavenging properties of A1 M makes it able to protect cells from the damaged incurred by heme.

[0008] As described in WO 2010 / 006809 and WO 2017 / 158181 , the following residues were identified as being important for the anti-oxidative effect of A1 M: Y22, C34, K69, K92, K118, K130, Y132, L180, 1181 , P182, and R183.

[0009] Recombinant human versions of A1 M (rA1 M, rhA1 M) have been developed for pharmaceutical evaluation (WO 2017 / 158181 ). Several variants of rA1 M that have modified amino acid sequences but retain the same biological effects have been developed and evaluated. Recombinant human A1 M (rA1 M) has been shown to be fully functional in comparison with endogenous A1 M derived from human plasma or urine.

[0010] Although the proteins can be expressed in an E. coli system, they lack glycosylation and display less solubility and lower stability compared to human A1 M purified from plasma. It is also envisaged that A1 M or rA1 M or mutants thereof must be administered parenterally in order to have the desired therapeutic effect, and as recombinant proteins lacking glycosylation, have a high risk of unwanted immunogenicity responses. However, it would be advantageous if it is possible to provide A1 M-mimicking peptides that are easy to prepare, that are of a size that makes it feasible to formulate pharmaceutical compositions, and with suitable physico-chemical and / or pharmacokinetic and / or biodistribution properties.

[0011] A1 M and variants thereof have shown great potential for the treatment of a collection of diseases and disorders (WO 2016 / 135214, WO 2017 / 158181 , and WO 2019 / 086569).

[0012] Thus, there is a need for developing peptides with the desired properties of A1 M, but which have a significantly smaller size. Such peptides would afford chronic dosing potential via a subcutaneous or nonparental route, lower risk of immunogenicity compared to full length complex protein, and through chemical synthesis provide a reduced cost of goods compared to E.coli produced recombinant proteins.

[0013] Summary

[0014] As outlined above, A1 M and variants thereof have shown great potential as a therapeutic biologic drug. However, certain disadvantages are associated with the use of proteins as active pharmaceutical ingredients (APIs), such as for instance the complexity of producing the proteins, as well as their stability / shelf-life. Accordingly, there’s an unmet need for novel APIs exhibiting therapeutic activity similar to A1 M, and having differential characteristics, e.g. regarding pharmacokinetic profile, tissues distribution, or by allowing for different administration routes with reduced potential for immunogenicity.

[0015] The present inventors have found that specific fragments of A1 M maintain the therapeutic activity of native A1 M, provided they fulfil the structural requirements as outlined herein. While it has been previously reported that several residues are important for the function of A1 M as outlined herein above, the present inventors have found that only a select few structural features appear to be paramount to the function of A1 M. Accordingly, the present inventors herein provide for polypeptides comprising one or more of said structural features, wherein the polypeptides have A1 M activity. The polypeptides disclosed herein are substantially shorter than A1 M and variants thereof previously reported. Accordingly, production and handling of the polypeptides is comparatively more feasible. Polypeptides may also be lyophilised, which many proteins cannot. It is further contemplated that the peptides have improved properties over A1 M and some of the previously reported A1 M variants, such as improved shelf life / stability. The disclosed polypeptides may also be administered via routes not viable for A1 M. The A1 M fragments disclosed herein are also of a length which permits for administration routes which are not feasible for the administration of full-length A1 M and variants thereof. The administration route can for example be oral, subcutaneous, or topical.

[0016] One aspect of the present disclosure provides for an agent comprising a polypeptide consisting of: a. 12 to 80 amino acid residues, wherein the polypeptide comprises the amino acid sequence GSTCPWLKKIX1(SEQ ID NO: 20) wherein X1is M, K, R, or Nle, or b. 30 to 80 amino acid residues, wherein the polypeptide comprises the amino acid sequence GATEAEISMTSTX2WRKGVCEETSGAYEKTD (SEQ ID NO: 21 ) wherein X2is H, R, or K.

[0017] One aspect of the present disclosure provides for an agent comprising a polypeptide consisting of 10 to 80 amino acid residues, wherein the polypeptide comprises a fragment of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 28, or SEQ ID NO: 29, wherein:

[0018] X3is selected from D, N, or G;

[0019] X4is selected from M, K, R, or Nle;

[0020] X5is selected from H, R, or K;

[0021] X6is selected from D, N, or E; and X7is selected from M or Nle; said fragment being at least 10 amino acid residues in length, wherein the fragment comprises an amino acid residue corresponding to C34 or C72 of SEQ ID NO: 30.

[0022] Polypeptides falling within this definition are shown herein to possess anti-oxidant activity. The polypeptides are also shown to protect cells against heme-induced cell death.

[0023] One aspect of the disclosure provides for an agent comprising the polypeptide of the disclosure and a further moiety.

[0024] One aspect of the present disclosure provides for an agent comprising a polypeptide consisting of a. 12 to 80 amino acid residues, wherein the polypeptide comprises the amino acid sequence GSTCPWLKKIX1(SEQ ID NO: 20) wherein X1is M, K, R, or Nle, or b. 30 to 80 amino acid residues, wherein the polypeptide comprises the amino acid sequence GATEAEISMTSTX2WRKGVCEETSGAYEKTD (SEQ ID NO: 21 ) wherein X2is H, R, or K; or

[0025] 10 to 80 amino acid residues, wherein the polypeptide comprises a fragment of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 28, or SEQ ID NO: 29, wherein:

[0026] X3is selected from D, N, or G;

[0027] X4is selected from M, K, R, or Nle;

[0028] X5is selected from H, R, or K;

[0029] X6is selected from D, N, or E;

[0030] X7is selected from M or Nle; said fragment being at least 10 amino acid residues in length, wherein the fragment comprises an amino acid residue corresponding to C34 or C72 of SEQ ID NO: 30; for use in the treatment or prophylaxis of a disease or disorder. One aspect of the present disclosure provides for a method of treating a disease or disorder comprising administering a therapeutically effective amount of the agent disclosed herein.

[0031] One aspect of the present disclosure provides for a use of the agent disclosed herein in the manufacture of a medicament for treatment of a disease or disorder.

[0032] Description of Drawings

[0033] Figure 1 : Cytochrome c reduction profiles for RMC-035 and GTI-1 to GTI-15.

[0034] Concentrations given in pM. Corresponding 4PL regression curves overlaid. GTI-1 to GTI-4 and GTI-6 to GTI-14 all show efficacy compared to RMC-035. In particular, the polypeptides show better than 30 % of the efficacy of RMC-035. Furthermore, EC50 values were found to be potent, with GTI-6 and GTI-11 to GTI-14 being especially potent. Negative controls GTI-5 and GTI-15 provided low efficacies, specifically under 30 % of the efficacy of RMC-035.

[0035] Figure 2: Cytochrome c reduction efficacy of RMC-035 and polypeptides GTI-1 to GTI- 15 given in % of the cytochrome c reduction efficacy of RMC-035.

[0036] Figure 3: EC50 values obtained in cytochrome c reduction assay for RMC-035 and GRI- 1 to GTI-15. Values which could not be obtained from the fit are shown as n.a.

[0037] Figure 4: RBC lysis profiles for RMC-035 and GTI-1 to GTI-15 showing LDH release. Concentrations given in pM. Corresponding 4PL regression curved overlaid. RMC-035, GTI-1 to GTI-4 and GTI-6 to GTI-12 all showed a decrease in absorption as the concentration of polypeptide increased. GTI-5 and GTI-15 showed no change in absorption, even at high polypeptide concentration.

[0038] Figure 5: RBC lysis profiles for RMC-035 and GTI-1 to GTI-15 showing haemoglobin release. Concentrations given in pM. Corresponding 4PL regression curves overlaid. RMC-035, GTI-1 to GTI-4 and GTI-6 to GTI-14 all showed a decrease in absorption of haemoglobin as the concentration of polypeptide increased, indicating that these polypeptides were capable of preventing RBC lysis. GTI-5 and GTI-15 showed no change in absorption, even at high polypeptide concentration. Figure 6: Synthetic peptides of human A1 M. (A) Amino acid sequences of two synthetic peptides covering part of human A1 M. The peptides were numbered and given names based on the first three amino acids followed by the number of amino acids. (B) Alignment of the two A1 M-peptides with the full-length amino acid sequence of human A1 M (Kaumeyer et al., 1986).

[0039] Figure 7: Localization of A1 M-peptides in the three-dimensional structure of human A1 M. The crystal structure of human A1 M (Meining and Skerra, 2012) is visualized using LICSF Chimera free software, version 1.10.2. The backbone of peptide ENF-46 (dark grey), the omega loopl (mid-grey helix), and the side-group of Cys34 (spacefilling atom model) are indicated.

[0040] Figure 8: SDS-PAGE of full-length A1 M and peptide ENF-46. Two micrograms of A1 M- wt, A1 M-035, and ten micrograms of peptide ENF-46 were incubated with sample buffer with or without p-mercaptoethanol, boiled for 1 min and separated on a 4-20% gradient polyacrylamide gel and finally stained with Coommassie Brilliant Blue.

[0041] Figure 9: Native PAGE of full-length A1 M and peptide ENF-46. Two micrograms of A1 M-wt or A1 M-035, or ten micrograms of peptide ENF-46 were incubated alone or with four-fold molar excess heme, and then separated by non-denaturing (native) PAGE. The gels were stained with Coommassie (left) or imaged by tryptophan fluorescence in a BioRad ChemiDoc equipment (right).

[0042] Figure 10: Reduction of cytochrome c by A1 M-wt and peptide ENF-46. Absorbance spectra of 100 pM cytochrome c in 20 mM Tris-HCI, pH 8, 0.15 M NaCI + 100 pM NADPH, with or without either 10 pM A1 M-wt or 20 pM peptide ENF-46, incubated 20 min at room-temperature. Buffer only was used as blank.

[0043] Figure 11 . Figure 11 A. Protection of cells against heme-induced cell death by A1 M and A1 M-peptides. (A) K562 cells, seeded at 1 x105cells / well in a 96-well microtiter plate, were incubated at 37°C with 100 pM heme in the presence of a dilution series of either ovalbumin (control), A1 M-wt, A1 M-035 or SRI-36 or ovalbumin for 1 hour. Incubations were performed in quadruplicates. Cell death was monitored as release of LDH into the medium, subtracting the LDH-value from live cells. Mean (n=4) ± SD is shown and statistical significances were calculated with ANOVA test and Dunett’s corrections for multiple comparisons. Figure 1 1 B-C. HK-2 cells were seeded at 5x104cells / well and incubated for 2 hours at 37°C with heme, A1 M-035, ENF-46 and / or SWT- 21 (control peptide) at concentrations shown in the figure. Incubations were performed in quadruplicates. After incubation, cells were analyzed for cell viability using WST-1 assay. Mean (n=4) ± SD is shown and statistical significances were calculated with ANOVA test and Tukey’s corrections for multiple comparisons. Figure 1 1 D-E. (D) HepG2 cells were seeded at 5x104 cells / well and incubated for 1 hour at 37°C with heme, A1 M-035 or ENF-46 at concentrations shown in the figure. Incubations were performed in quadruplicates. Cell death was monitored as release of LDH into the medium, subtracting the LDH-value from live cells. Mean (n=4) ± SD is shown and statistical significances were calculated with ANOVA test and Tukey’s corrections for multiple comparisons. (E) Red blood cells (RBC) were suspended at 1% (v / v) and incubated under rotation for 3 hours (except control, 0 hour) at room temperature with heme, A1 M-035 and / or ENF-46 at concentrations shown in the figure. Incubations were performed in triplicates. Cell death was monitored as release of LDH into the medium. Mean (n=3) ± SD is shown and statistical significances were calculated with ANOVA test and Tukey’s corrections for multiple comparisons. *p < 0.05, **p < 0.01 , ***p < 0.001.

[0044] Figure 12. Human Kidney-2 (HK-2) proximal tubule cell line viability profiles for RMC- 035 and GTI-1 to GTI-15, GTI-24, GTI-25, GTI-29 to GTI-31 , GTI-46, GTI-50, GTI-61 - 64, GTI-73, showing lactase dehydrogenase (LDH) release. Concentrations given in pM. Corresponding 4PL regression curves overlaid. RMC-035, and all peptides tested except GTI-5, GTI-7, GTI-5, GTI-7, GTI-13, GTI-14, and GTI-15, showed a decrease in LDH absorption as the concentration of polypeptide increased, demonstrating the capacity of the polypeptides to inhibit a heme-induced reduction in cell viability. GTI-5 and GTI-15 showed no change in absorption, even at high polypeptide concentration.

[0045] Figure 13. HO-1 mRNA expression as determined by RT-qPCR.

[0046] Figure 14. Serum creatinine and blood urea nitrogen (BUN) following uni-nephrectomy and renal ischemia / reperfusion injury and treatment with peptide GTI-1 1 . Serum creatinine and BUN measured at Baseline pre-surgery, 1 , 2, 3 and 5 days following unilateral nephrectomy and 40 min unilateral renal pedicle clamping in rats. n=7 / 8, values are presented as mean±SEM. Differences between GTI-1 1 treated animals versus vehicle were analysed using the Mann-Whitney ll-test. * P< 0.05; ** P <0.01 , *** P <0.001.

[0047] Figure 15. Rat pCreatinine (mg / dL) and blood urea nitrogen (BUN, mg / dL) 1 and 3- days post-uni-nephrectomy and ischemia reperfusion injury and treatment with either saline vehicle or peptide GTI-2 by IV or SC administration. Values are presented as mean±SEM. Differences between GTI-2 treated animals versus vehicle were analysed using the Mann-Whitney U-test. * P < 0.05; ** P <0.01 .

[0048] Figure 16. Rat glomerular filtration rate (GFR) 3-days post-uni-nephrectomy and ischemia reperfusion injury and treatment with either saline vehicle or peptide GTI-2 by IV or SC administration. Values are presented as mean±SEM. Differences between GTI-2 treated animals versus vehicle were analysed using the Mann-Whitney U-test. * P < 0.05; “ P <0.01.

[0049] Figure 17. Mouse pCreatinine (pCreatinine, mg / dL), blood urea nitrogen (BUN, mg / dL) and aspartate amino transferase (AST, nmol / mL / min) at baseline pre-LPS, 8 and 24 hours post LPS and treatment with either saline vehicle or peptide GTI-2 by IV administration. Values are presented as mean±SEM. Differences between GTI-2 treated animals versus vehicle were analysed using the Mann-Whitney U-test. * P < 0.05; *** P <0.001 ; **** P <0.0001 .

[0050] Figure 18. Mouse pCreatinine (mg / dL), and blood urea nitrogen (BUN, mg / dL) at baseline and day 4 post-cisplatin-kidney injury and treatment with either PBS vehicle or peptide GTI-2 or GTI-86 by IV or SC administration. Values are presented as mean±SEM. Differences between GTI-2 treated animals versus vehicle were analysed using the Mann-Whitney U-test. ** P <0.01 ; *** P <0.001 .

[0051] Figure 19. Mouse pCreatinine and urine albumin / creatinine ratios (UACR, mg / mmol) at Day 0 (25 days following STZ treatment), and Days 14 and 28 of treatment with A1 M tool peptide (RMC-035) by SC administration. Values are presented as mean±SEM. Differences between vehicle, STZ and A1 M tool peptide treated animals versus vehicle were analysed using the Mann-Whitney U-test. *P<0.05, ** P <0.01 , ***P<0.001. Figure 20. Adriamycin treatment significantly increases pCreatinine, BUN and UACR levels in Adriamycin-treated versus vehicle treated mice at both 9- and 14-days post Adriamycin challenge (Figure 20). The Adriamycin-induced increases in pCreatinine (Figure 20A), BUN (Figure 20B) and UACR (Figure 20C) were all significantly inhibited by daily GTI-86 treatment in a dose dependent manner.* P<0.05 and **P<0.01 .

[0052] Figure 21 . A1 M-035 (Figure 21 A) and peptides GTI-86, GTI-111 and GTI-115 (Figures 21 B-D) bind free heme as indicated by fluorescence microscopy where a shift in absorbance is observed between free heme and heme complexed with either A1 M-035 or peptides. E: GTI-86 binds to heme as evidenced by increase absorbance peak at 383 nm. GTI-86 also increased reductase activity in a dose-dependent manner as evidenced by the absorbance between 400 and 470 nm.

[0053] Detailed description

[0054] Definitions

[0055] As used herein, the phrase “consisting of between X and Y amino acid residues” is taken to mean a polypeptide consisting of a number of amino acid residues X to Y both thresholds included, i.e. the polypeptide may consist of X amino acid residues, Y amino acid residues, or a number of amino acid residues falling between X and Y, but it cannot consist of more than Y amino acid residues. By way of example, a polypeptide comprising of 3 to 6 amino acid residues consists of exactly 3, 4, 5, or 6 amino acid residues.

[0056] As used herein "fragment", when used in reference to a reference polypeptide, is a polypeptide having an amino acid sequence that is the same as part of but not all of the amino acid sequence of the reference polypeptide.

[0057] By “variant” is meant that the peptide fragment does not share 100 % amino acid sequence identity with the reference sequence, i.e. one or more amino acid residues of the peptide fragment is mutated relative to the reference sequence and / or there is one or more gaps (i.e. a deletion) of one or more amino acid residues relative to the reference sequence. For example, the peptide fragment may have at least 80 % sequence identity to the reference sequence. By a “variant comprising X contiguous amino acid residues of a reference sequence” is meant that the variant comprises a sequence of X amino acid residues which have 100 % sequence identity to the reference sequence.

[0058] As used herein, “polypeptide fragment” “A1 M fragment” “RMC-035 fragment” are used interchangeably.

[0059] Numbering of amino acid residues throughout the document refers to SEQ ID 30; when describing other sequences, a person skilled in the art will know how to identify the amino acid residues corresponding to the amino acid residues in SEQ ID NO: 30 based on the context and the sequence overlap.

[0060] As used herein, the compound GTI-1 corresponds to a polypeptide having SEQ ID NO: 1 . As used herein, the compound GTI-2 corresponds to a polypeptide having SEQ ID NO: 2. As used herein, the compound GTI-3 corresponds to a polypeptide having SEQ ID NO: 3. As used herein, the compound GTI-4 corresponds to a polypeptide having SEQ ID NO: 4. As used herein, the compound GTI-5 corresponds to a polypeptide having SEQ ID NO: 5. As used herein, the compound GTI-6 corresponds to a polypeptide having SEQ ID NO: 6. As used herein, the compound GTI-7 corresponds to a polypeptide having SEQ ID NO: 7. As used herein, the compound GTI-8 corresponds to a polypeptide having SEQ ID NO: 8. As used herein, the compound GTI-9 corresponds to a polypeptide having SEQ ID NO: 9. As used herein, the compound GTI-10 corresponds to a polypeptide having SEQ ID NO: 10. As used herein, the compound GTI-11 corresponds to a polypeptide having SEQ ID NO: 11 . As used herein, the compound GTI-12 corresponds to a polypeptide having SEQ ID NO: 12. As used herein, the compound GTI-13 corresponds to a polypeptide having SEQ ID NO: 13. As used herein, the compound GTI-14 corresponds to a polypeptide having SEQ ID NO: 14. As used herein, the compound GTI-15 corresponds to a polypeptide having SEQ ID NO: 15. As used herein, the compound RMC-035 (A1 M-035) corresponds to a polypeptide having SEQ ID NO: 16.

[0061] As used herein the terms "treatment" or" treating" is an approach for obtaining beneficial or desired results including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease or disorder, stabilized (i.e., not worsening) state of disease or disorder, prevention of the disease or disorder, delay or slowing of disease or disorder progression, amelioration or palliation of the disease state, and remission (whether partial or total) whether detectable or undetectable.

[0062] As used herein the term “A1 M activity” refers to the property of a compound to act similarly to A1 M. Specifically, the compound may be an anti-oxidant, it may bind heme, it may be capable of reducing cytochrome c, or exhibit any other physiological or therapeutic effect that A1 M exhibits.

[0063] By “CLIPS” is meant a modification with 1 ,3-bis(bromomethyl)benzene. Nucleophilic moieties of amino acids can react on the bromomethyl-carbon to effect a substitution of Br with the nucleophilic moiety of the amino acid.

[0064] Unless otherwise specified, the peptides disclosed herein have an N-terminal acetylation and C-terminal amidation.

[0065] The names “A1 M-035” and “RMC-035” are used interchangeably for the same protein.

[0066] As used herein, "oxidative stress" means an imbalance between the production and manifestation of excessive levels of molecular oxygen or reactive oxygen species (ROS) and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage via endogenous antioxidant systems. Disturbances in the normal redox state of tissues can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids, and DNA via mechanisms including lipid peroxidation, protein oxidation and aggregation and DNA damage. Some reactive oxidative species can even act as messengers through a phenomenon called redox signalling.

[0067] The term "haematuria" as used herein is defined as presence of red blood cells (RBCs) in the urine, wherein microscopic analysis demonstrates that >3 RBCs per high-power field (HPF) are present in a urine sample or urinalysis demonstrates a positive dipstick test. Haematuria can be gross (visible in the blood) or microscopic (detection of blood on urinalysis or urine microscopy). Haematuria can be intermittent or persistent. Other methods to evaluate haematuria include renal parameters (such as glomerular filtration rate; GFR, serum creatinine, urea nitrogen; BUN), imaging (ultrasound, CT, MRI), cystoscopy, urine cytology and / or kidney biopsy.

[0068] The term "glomerular haematuria" as used herein is defined as haematuria of glomerular aetiology.

[0069] The term “glomerulonephritis” as used herein is defined as inflammation of the glomerulus or glomeruli and / or small blood vessels of the kidney.

[0070] As used herein, the term "acute kidney injury" (AKI), previously referred to as "acute renal failure" (ARF), refers to an acute clinical syndrome characterised by rapid decline in renal functions, which is caused by a number of factors such as a reduction in renal blood flow, glomerulonephritis, use of nephrotoxic antibiotics and anticancer agents. Oxidative stress and inflammation are predominant pathogenic mediators of AKI. The duration of AKI can last for up to 7 days.

[0071] As used herein, the term "acute kidney disease" (AKD), refers to an AKI which lasts 7 to 90 days.

[0072] As used herein, the term "chronic kidney disease" (CKD) refers the presence of kidney damage or an estimated glomerular filtration rate (eGFR) less than 60 ml / min per 1 .73 square meters, persisting for 90 days or more. It is a state of progressive loss of kidney function ultimately resulting in the need for renal replacement therapy (dialysis or transplantation). CKD has its general meaning in the art and is used to classify numerous conditions that affect the kidney, destruction of the renal parenchyma and the loss of functional nephrons or glomeruli. Examples of aetiology of CKD include, but are not limited to, cardiovascular diseases, hypertension, diabetes, glomerulonephritis, polycystic kidney diseases, and recipients of a kidney graft.

[0073] Polypeptide fraoments and variants thereof

[0074] The present invention relates to novel polypeptides which are fragments of A1 M or variants thereof. An object of the present disclosure is the provision of A1 M fragments having A1 M activity. A1 M and variants thereof, such as disclosed RMC-035, have effective therapy against diseases and disorders related to heme and oxidative stress. The mechanism of A1 M treatment involve anti-oxidant, heme-binding, reductase activity, and mitochondrial protection. The following residues have been reported as being important for the function of A1 M: Y22, C34, K69, K92, K1 18, K130, Y132, L180, 1181 , P182, and R183. However, the present inventors have discovered that some A1 M fragments, which do not comprise the majority of these residues, have maintained A1 M activity.

[0075] The present inventors have surprisingly discovered that certain peptide fragments of A1 M maintain the A1 M activity. Specifically, the inventors found A1 M activity in A1 M fragments designed to comprise an 1 1 -aa fragment corresponding to residues G31 to M41 and / or a 30-aa fragment corresponding to residues G54 to D83 of SEQ ID NO: 30. Specifically, the fragments may comprise variable positions as disclosed herein. The present inventors have also found that further non-native modifications can be introduced to said A1 M fragments to further improve the properties, e.g. enhance potency and / or stability etc.

[0076] One embodiment of the present disclosure provides for an agent comprising the polypeptide as disclosed herein. In one embodiment, the agent consists of the polypeptide disclosed herein.

[0077] One embodiment of the present disclosure provides for an agent comprising a polypeptide consisting of a. 12 to 80 amino acid residues, wherein the polypeptide comprises the amino acid sequence GSTCPWLKKIX1(SEQ ID NO: 20) wherein X1is M, K, R, or Nle, or b. 30 to 80 amino acid residues, wherein the polypeptide comprises the amino acid sequence GATEAEISMTSTX2WRKGVCEETSGAYEKTD (SEQ ID NO: 21 ) wherein X2is H, R, or K.

[0078] One embodiment of the present disclosure provides for an agent comprising a polypeptide consisting of 10 to 80 amino acid residues, wherein the polypeptide comprises a fragment of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 28, or SEQ ID NO: 29, wherein:

[0079] X3is selected from D, N, or G;

[0080] X4is selected from M, K, R, or Nle;

[0081] X5is selected from H, R, or K; X6is selected from D, N, or E; and

[0082] X7is selected from M or Nle; said fragment being at least 10 amino acid residues in length, wherein the fragment comprises an amino acid residue corresponding to C34 or C72 of SEQ ID NO: 30.

[0083] One embodiment of the present disclosure provides for an agent comprising a polypeptide consisting of at least 10 amino acid residues, wherein the polypeptide comprises a fragment of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 28, or SEQ ID NO: 29, wherein:

[0084] X3is selected from D, N, or G;

[0085] X4is selected from M, K, R, or Nle;

[0086] X5is selected from H, R, or K;

[0087] X6is selected from D, N, or E; and

[0088] X7is selected from M or Nle, said fragment being at least 10 amino acid residues in length, wherein the fragment comprises an amino acid residue corresponding to C34 or C72 of SEQ ID NO: 30.

[0089] In one embodiment the polypeptide comprises the amino acid sequence GSTCPWLKKIX1(SEQ ID NO: 20) wherein X1is M, K, R, or Nle.

[0090] In one embodiment the polypeptide comprises the amino acid sequence GATEAEISMTSTX2WRKGVCEETSGAYEKTD (SEQ ID NO: 21) wherein X2is H, R, or K.

[0091] In one embodiment the polypeptide comprises the amino acid sequence GSTCPWLKKIM (SEQ ID NO: 22). In one embodiment the polypeptide comprises the amino acid sequence GSTCPWLKKIK (SEQ ID NO: 23). In one embodiment the polypeptide comprises the amino acid sequence GSTCPWLKKIR (SEQ ID NO: 24). In a preferred embodiment the present disclosure, the polypeptide comprises the amino acid sequence GSTCPWLKKIM (SEQ ID NO: 22). In a preferred embodiment the present disclosure, the polypeptide comprises the amino acid sequence GSTCPWLKKI[Nle] (SEQ ID NO: 145). In one embodiment the polypeptide comprises the amino acid sequence GATEAEISMTSTHWRKGVCEETSGAYEKTD (SEQ ID NO: 25). In one embodiment the polypeptide comprises the amino acid sequence GATEAEISMTSTRWRKGVCEETSGAYEKTD (SEQ ID NO: 26). In one embodiment the polypeptide comprises the amino acid sequence GATEAEISMTSTKWRKGVCEETSGAYEKTD (SEQ ID NO: 27).

[0092] In one embodiment of the present disclosure, the polypeptide is a fragment of SEQ ID NO: 28. In one embodiment of the present disclosure, the polypeptide is a fragment of SEQ ID NO: 29. The polypeptide sequence corresponding to SEQ ID NO: 28 and SEQ ID NO: 29 comprises variable residues. Should the polypeptide fragment as disclosed herein comprise two or more of the variable residues of SEQ ID NO: 28 or SEQ ID NO: 29, any combination variable residues as defined herein are contemplated to form part of the disclosure.

[0093] In one embodiment of the disclosure, the polypeptide is a fragment of SEQ ID NO: 28 or SEQ ID NO: 29 wherein X3is selected from the group consisting of D, N, and G. In one embodiment of the disclosure, the polypeptide is a fragment of SEQ ID NO: 28 or SEQ ID NO: 29 wherein X4is selected from the group consisting of M, K, R, and Nle. In one embodiment of the disclosure, the polypeptide is a fragment of SEQ ID NO: 28 or SEQ ID NO: 29 wherein X5is selected from the group consisting of H, R, and K. In one embodiment of the disclosure, the polypeptide is a fragment of SEQ ID NO: 28 or SEQ ID NO: 29 wherein X6is selected from the group consisting of D, N, and E. In one embodiment of the disclosure, the polypeptide is a fragment of SEQ ID NO: 28 or SEQ ID NO: 29 wherein X7is selected from the group consisting of M and Nle. In one embodiment of the disclosure, the polypeptide is a fragment of SEQ ID NO: 28 or SEQ ID NO: 29 wherein X3is selected from the group consisting of D, N, and G, X4is selected from the group consisting of M, K, and R, X5is selected from the group consisting of H, R, and K, X6is selected from the group consisting of D, N, and E, and X7is selected from the group consisting of M and Nle.

[0094] In one embodiment of the present disclosure, the polypeptide is a fragment of SEQ ID NO: 16. In one embodiment of the present disclosure, the polypeptide is a fragment of SEQ ID NO: 17. In one embodiment of the present disclosure, the polypeptide is a fragment of SEQ ID NO: 18. In one embodiment of the present disclosure, the polypeptide is a fragment of SEQ ID NO: 19. It is contemplated that certain residues of the polypeptides of the disclosure may be replaced with chemically similar residues, i.e. the residues may be replaced with other residues following the principle of “conservative substitution”. In one embodiment of the present disclosure, the polypeptides of the disclosure may differ from the polypeptides defined herein by the presence of 1 conservative amino acid substitution. In one embodiment of the present disclosure, the polypeptides of the disclosure may differ from the polypeptides defined herein by the presence of 2 conservative amino acid substitutions. In one embodiment of the present disclosure, the polypeptides of the disclosure may differ from the polypeptides defined herein by the presence of 3 conservative amino acid substitution. In one embodiment of the present disclosure, the polypeptides of the disclosure may differ from the polypeptides defined herein by the presence of 4 conservative amino acid substitutions. In one embodiment of the present disclosure, the polypeptides of the disclosure may differ from the polypeptides defined herein by the presence of 5 conservative amino acid substitutions. Those of skill in the art will recognize that individual additions deletions, insertions and / or substitutions to an amino acid sequence that alter a single amino acid or a small percentage of amino acids results in the conservation of the properties of the original amino acid side-chain; it is thus referred to as "conservative substitution" or "conservative modification", wherein the alteration of a protein results in a protein with similar functions. Conservative substitution tables providing functionally similar amino acids are well known in the art. Examples of properties of amino acid side chains are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side chains having the following functional groups or characteristics in common: an aliphatic side-chain (G, A, V, L, I, P); a hydroxyl group containing sidechain (S, T, Y); a sulfur atom containing side-chain (C, M); a carboxylic acid and amide containing side-chain (D, N, E, Q); a base containing side-chain (R, K, H); and an aromatic group containing side-chain (H, F, Y, W). In addition, the following eight groups each contain amino acids that are conservative substitutions for one another: 1 ) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Aspargine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M). However, in a preferred embodiment of the disclosure, the cysteine moieties as defined herein, i.e. C34 and C72, are not substituted. It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and the like. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within + 2 is preferred, those within + 1 are particularly preferred, and those within + 0.5 are even more particularly preferred.

[0095] In one embodiment of the present disclosure, the polypeptide consists of 12 to 80 amino acid residues. In a further embodiment of the disclosure, the polypeptide consists of 12 to 80 amino acid residues and comprises the amino acid sequence GSTCPWLKKIX1(SEQ ID NO: 20) wherein X1is as defined herein.

[0096] In one embodiment of the present disclosure, the polypeptide consists of 30 to 80 amino acid residues. In a further embodiment of the present disclosure, the polypeptide consists of 30 to 80 amino acid residues and comprises the amino acid sequence GATEAEISMTSTX2WRKGVCEETSGAYEKTD (SEQ ID NO: 21) wherein X2is as defined herein.

[0097] In one embodiment of the present disclosure, the polypeptide consists of at least 12 amino acid residues. In one embodiment, the polypeptide consists of at least 13 amino acid residues. In one embodiment, the polypeptide consists of at least 14 amino acid residues. In one embodiment, the polypeptide consists of at least 15 amino acid residues, such as at least 16, such as at least 17, such as at least 18, such as at least 19, such as at least 20, such as at least 21 , such as at least 22, such as at least 23, such as at least 24 amino acid residues.

[0098] In one embodiment of the present disclosure, the polypeptide consists of no more than 170 amino acid residues, such as no more than 160, such as no more than 150, such as no more than 140, such as no more than 130, such as no more than 120, such as no more than 110, such as no more than 100, such as no more than 90 amino acid residues. In one embodiment of the present disclosure, the polypeptide consists of no more than 80 amino acid residues, such as no more than 79, such as no more than 78, such as no more than 77, such as no more than 76, such as no more than 75, such as no more than 74, such as no more than 73, such as no more than 72, such as no more than 71 , such as no more than 70, such as no more than 69, such as no more than 68, such as no more than 67, such as no more than 66, such as no more than 65, such as no more than 64, such as no more than 63, such as no more than 62, such as no more than 61 , such as no more than 60, such as no more than 59, such as no more than 58, such as no more than 57, such as no more than 56, such as no more than 55, such as no more than 54, such as no more than 53, such as no more than 52, such as no more than 51 , such as no more than 50, such as no more than 49, such as no more than 48, such as no more than 47, such as no more than 46, such as no more than 45, such as no more than 44, such as no more than 43, such as no more than 42, such as no more than 41 , such as no more than 40, such as no more than 39, such as no more than 38, such as no more than 37, such as no more than 36, such as no more than 35 amino acid residues.

[0099] In one embodiment of the present disclosure, the polypeptide consists of 12 to 15 amino acid residues, such as 12 to 15, such as 15 to 20, such as 20 to 25, such as 25 to 30, such as 30 to 35, such as 35 to 40, such as 40 to 45, such as 45 to 50, such as 50 to 55, such as 55 to 60, such as 60 to 65, such as 65 to 70, such as 70 to 75, such as 75 to 80 amino acid residues.

[0100] In one embodiment of the present disclosure, the polypeptide comprises at least 4 contiguous amino acid residues of the reference sequence, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10, such as at least 11 , such as at least 12 contiguous amino acid residues.

[0101] Human A1 M (SEQ ID NO: 30) is a 183 residue protein. One embodiment of the present disclosure provides for an agent comprising a fragment of human A1 M. In one embodiment of the present disclosure, the fragment consists of a sequence corresponding to residues residing towards the N-terminal end of A1 M or a variant thereof. In one embodiment, the fragment consists of a sequence corresponding to positions 1 to 180 of SEQ ID NO: 30. In one embodiment, the fragment consists of a sequence corresponding to positions 1 to 170 of SEQ ID NO: 30. In one embodiment, the fragment consists of a sequence corresponding to positions 1 to 160 of SEQ ID NO: 30. In one embodiment, the fragment consists of a sequence corresponding to positions 1 to 150 of SEQ ID NO: 30. In one embodiment, the fragment consists of a sequence corresponding to positions 1 to 140 of SEQ ID NO: 30. In one embodiment, the fragment consists of a sequence corresponding to positions 1 to 130 of SEQ ID NO: 30. In one embodiment, the fragment consists of a sequence corresponding to positions 1 to 120 of SEQ ID NO: 30. In one embodiment, the fragment consists of a sequence corresponding to positions 1 to 110 of SEQ ID NO: 30. In one embodiment, the fragment consists of a sequence corresponding to positions 1 to 100 of SEQ ID NO: 30. In one embodiment, the fragment consists of a sequence corresponding to positions 1 to 90 of SEQ ID NO: 30.

[0102] In a preferred embodiment of the present disclosure, the polypeptide comprises or consists of an amino acid sequence selected from the group consisting of i. DDDDKQVQENFDISRIYGKWYNLAIGSTCPWLKKIMDRMTVSTLVLGEGAT (SEQ ID NO: 1), ii. DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGAT (SEQ ID NO: 2), ill. DDKGPVPTPPDNIQVQENFDISRIYGKWYNLAIGSTCPWLKKIM (SEQ ID

[0103] NO: 3), iv. KGSTCPWLKKIMDRMTVSTLVLGEGAT (SEQ ID NO: 4), v. GSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTHWRKGVCEETSGAYEK (SEQ ID NO: 6), vi. DDDDKGPVPTPPDNIQVQENFDISRIYGKWYNLAIGSTCPWLKKIMDRM (SEQ ID NO: 7), vii. GSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTHWRKGVCEET (SEQ ID NO: 8), viii. GSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTHWRK (SEQ ID NO: 9), ix. GSTCPWLKKIMDRMTVSTLVLGEGATEAEISMT (SEQ ID NO: 10), x. GSTCPWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 11 ), xi. NLAIGSTCPWLKKIMDR (SEQ ID NO: 12), xii. GEGATEAEISMTSTHWRKGVCEETSGAYEKTDTDG (SEQ ID NO: 13), and xiii. GATEAEISMTSTHWRKGVCEETSGAYEKTD (SEQ ID NO: 14). In a preferred embodiment of the present disclosure, the polypeptide comprises or consists of the amino acid sequence GSTCPWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 11 ).

[0104] Based on the results outlined in the examples herein below, it is contemplated that the cysteine residues corresponding to C34 and C72 are especially important for the function of the polypeptides disclosed herein. This consideration is based on the observations that i) polypeptide showing A1 M activity (i.e. cytochrome c reduction assay activity and / or RBC assay activity) comprises a residue or residues corresponding to C34 and / or C72 and ii) polypeptides not comprising a residue corresponding to C34 and / or C72 do not show A1 M activity. Accordingly, in a preferred embodiment of the present disclosure, the polypeptide comprises a residue corresponding to C34. In another preferred embodiment of the disclosure, the polypeptide comprises a residue corresponding to C72. In another preferred embodiment of the disclosure, the polypeptide comprises a residue corresponding to C34 and a residue corresponding to C72. One embodiment of the present disclosure provides for a polypeptide which i) is a fragment of A1 M, ii) comprises a free cysteine moiety, and iii) has A1 M activity.

[0105] The polypeptide of the present disclosure may be produced using known techniques, such as by solid phase synthesis or by production in a genetically engineered organism, i.e. by recombinant production.

[0106] The present disclosure provides a peptide having a length of from 10 to 92 amino acid residues, wherein the peptide comprises a fragment selected from position 1 to position 92 of human wt alpha-1 -microglobulin, A1 M, SEQ ID NO: 83: GPVPTPPDNI QVQENFNISR IYGKWYNLAI GSTCPWLKKI MDRMTVSTLV 1 -50 LGEGATEAEI SMTSTRWRKG VCEETSGAYE KTDTDGKFLY HK, wherein the fragment has a length of from 10 to 92 amino acid residues, and wherein the fragment includes one or more of Y22, C34, K69 or K92, where the positions refer to the positions in human wtA1 M (SEQ ID NO: 30). The fragment may be selected from position 1 to position 92 of human wtA1 M, and wherein the fragment has at least 50% identity with the corresponding fragment of human wtA1 M. The fragment may have a length of from 15 to 80 amino acid residues such as from 20 to 75 amino acid residues.The fragment may be selected from position 22 to position 92 of human wtA1M, SEQ ID NO: 84: YGKWYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGA YEKTDTDGKFLYHK, and then the fragment has a length of from 10 to 71 amino acid residues.

[0107] In one embodiment of the disclosure the fragment is selected from one of the following positions of human wt A1 M: i) Position 22 to position 62 (SEQ ID NO: 85), ii) Position 26 to position 62 (SEQ ID NO: 86), ill) Position 30 to position 62 (SEQ ID NO: 87), iv) position 31 to position 56 (SEQ ID NO: 88), v) position 26 to position 56 (SEQ ID NO: 89), vi) position 26 to position 92 (SEQ ID NO: 90), vii) position 30 to position 92 (SEQ ID NO: 91), or viii) position 31 to position 92 (SEQ ID NO: 92).

[0108] The peptides of the disclosure have a length of at the most 92 aa (amino acids), notably at the most 80 aa such as at the most 75 aa, at the most 71 aa, at the most 60 aa, at the most 55 aa, at the most 50 aa such as at the most 49 aa, at the most 48 aa, at the most 47 aa, at the most 46 aa, at the most 45 aa, at the most 44 aa, at the most 43 AA, at the most 42 aa, at the most 41 aa, at the most 40 aa, at the most 39 aa, at the most 38 aa, at the most 37 aa, at the most 36 aa, at the most 35 aa, at the most 34 aa, at the most 33 aa, at the most 32 aa, at the most 31 aa, at the most 30 aa, at the most 29 aa, at the most 28 aa or at the most 27 aa. In general, the minimal length of the peptides of the disclosure is 10 aa. In general, the length of the peptides of the disclosure is from 10 to 80 aa such as from 10 to 70 aa, from 10 to 60 aa or from 10 to 50 aa. The peptides mentioned in the examples herein have a length of from 30 to 50 aa such as from 35 to 50 aa.

[0109] A peptide of the present disclosure may comprise an amino acid sequence having a sequence identity of 50% or more to one of the following sequences of human wt A1 M: i) Position 22 to position 62, ii) Position 26 to position 62, ill) Position 30 to position 62, iv) position 31 to position 56, v) position 26 to position 56, vi) position 26 to position 92, vii) position 30 to position 92, or viii) position 31 to position 92.

[0110] A peptide of the disclosure may comprise an amino acid sequence corresponding to one of the following sequences of human wt A1 M: i) Position 22 to position 62, ii) Position 26 to position 62, ill) Position 30 to position 62, iv) position 31 to position 56, v) position 26 to position 56, vi) position 26 to position 92, vii) position 30 to position 92, or viii) position 31 to position 92.

[0111] Moreover, a peptide according to the disclosure may at the N-terminal end and or the C-terminal comprises an amino acid sequence, Z, having a length of from 1 to 20 amino acid residue. In one embodiment, such an amino acid sequence is a tag, i.e. a polypeptide segment that can be attached to the peptides disclosed herein to provide for further advantages, such as for purification, solubility, or detection of the peptide. The term "peptide tag" as used herein generally refers to a small peptide fragment which may or may not be designed or derived directly from the isopeptide protein.

[0112] Z may comprise: i) one or more lysine, K, residue(s), one or more glutamic acid, E, residue(s), one or more arginine, R, residue(s) and / or one or more aspartic acid, D, residue(s), ii) one or more hydrophobic amino acid residue(s), ill) one or more of histidine H, residue(s), one or more of glutamine, G, residue(s), and / or one or more of isoleucine, I residue(s), and / or at the N-terminal end Z is or comprises one or more of the following sequences: i) HHHHHHHHGGGGGIEGR (8H5GIEGR) (SEQ ID NO: 93), ii) HHHHHHHHDDDDK (8H4DK) (SEQ ID NO: 94), ill) HHHHHHDDDDK (6H4DK) (SEQ ID NO: 95), or iv) HHHHHHHH (8H) (SEQ ID NO: 96), and / or at the C-terminal end Z is or comprises one or more of the following sequences: i) RGIEGGGGGHHHHHHHH (RGIE5G8H) (SEQ ID NO: 97) ii) KDDDDHHHHHHHH (K4D8H) (SEQ ID NO: 98) ill) KDDDDHHHHHH (K4D6H) (SEQ ID NO: 99), or iv) HHHHHHHH (8H) (SEQ ID NO: 96),

[0113] A peptide according to the disclosure may comprises a sequence selected from:

[0114] Y-rn-K,

[0115] Y-F12-C, or

[0116] C-F13-K, wherein fl 1 has at least 50% sequence identity with SEQ ID NO: 100, which is GKWYNLAI GSTCPWLKKI MDRMTVSTLV

[0117] LGEGATEAEI SMTSTRWRKG VCEETSGAYE KTDTDGKFLY H (corresponding to pos 23-91 of wtA1 M)

[0118] FI2 has at least 50% sequence identity with SEQ ID NO: 101 , which is GKWYNLAI GST (corresponding to pos 23-33 of wtA1 M), and

[0119] FI3 has at least 50% sequence identity with SEQ ID NO: 102, which is

[0120] PWLKKI MDRMTVSTLV LGEGATEAEI SMTSTRWRKG VCEETSGAYE KTDTDGKFLY H (corresponding to pos 35-91 of wtA1 M).

[0121] In embodiments of the disclosure the peptide has at least 50% sequence identity with SEQ ID NO: 83.

[0122] As it appears from the examples herein, peptides of the disclosure include i) an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 103, which is SR IYGKWYNLAI GSTCPWLKKI MDRMTVSTLV LGEG (corresponding to SRI-36, pos 19-54 of wtA1 M), ii) an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 104, which is ENFNISR IYGKWYNLAI GSTCPWLKKI MDRMTVSTLV LGEGATEAE (corresponding to ENF-46, pos 14-59 of wtA1 M)

[0123] One embodiment of the disclosure is a polypeptide having a length of from 10 to 92 amino acid residues, wherein the peptide comprises a fragment selected from position 1 to position 92 of the human wt alpha-1 -microglobulin, A1 M, sequence defined in SEQ ID NO: 83. In one embodiment of the disclosure, the fragment has a length from 10 to 92 amino acid residues. In one embodiment of the disclosure, the fragment includes one or more of Y22, C34, K69 or K92, where the positions refer to the positions in human wtA1 M (SEQ ID NO: 30). One embodiment of the disclosure, the fragment is selected from position 1 to position 92 of human wtA1 M, and wherein the fragment has at least 50% identity with the corresponding fragment of human wtA1 M. One embodiment of the disclosure is a fragment having a length from 15 to 80 amino acid residues such as from 20 to 75 amino acid residues.

[0124] In one embodiment of the disclosure, the fragment is the sequence defined in SEQ ID NO 84, corresponding to position 22-92 of human wtA1 M. In one embodiment of the disclosure, the fragment is selected from the sequence defined in SEQ ID NO 84, wherein the fragment has a length from 10 to 71 amino acid residues.

[0125] In one embodiment of the disclosure, the fragment is defined in SEQ ID NO 85, corresponding to position 22-62 of human wtA1 M. In one embodiment of the disclosure, the fragment is defined in SEQ ID NO 86, corresponding to position 26-62 of human wtA1 M. In one embodiment of the disclosure, the fragment is defined in SEQ ID NO 87, corresponding to position 30-62 of human wtA1 M. In one embodiment of the disclosure, the fragment is defined in SEQ ID NO 88, corresponding to position 31 -56 of human wtA1 M. In one embodiment of the disclosure, the fragment is defined in SEQ ID NO 89, corresponding to position 26-56 of human wtA1 M. In one embodiment of the disclosure, the fragment is defined in SEQ ID NO 90, corresponding to position 26-92 of human wtA1 M. In one embodiment of the disclosure, the fragment is defined in SEQ ID NO 91 , corresponding to position 30-92 of human wtA1 M. In one embodiment of the disclosure, the fragment is defined in SEQ ID NO 92, corresponding to position 31 -92 of human wtA1 M.

[0126] In one embodiment of the disclosure the peptide has a length of at the most 92 aa (amino acids), notably at the most 80 aa such as at the most 75 aa, at the most 71 aa, at the most 60 aa, at the most 55 aa, at the most 50 aa such as at the most 49 aa, at the most 48 aa, at the most 47 aa, at the most 46 aa, at the most 45 aa, at the most 44 aa, at the most 43 AA, at the most 42 aa, at the most 41 aa, at the most 40 aa, at the most 39 aa, at the most 38 aa, at the most 37 aa, at the most 36 aa, at the most 35 aa, at the most 34 aa, at the most 33 aa, at the most 32 aa, at the most 31 aa, at the most 30 aa, at the most 29 aa, at the most 28 aa or at the most 27 aa. In one embodiment of the disclosure, the minimal length of the peptide is 10 aa. In one embodiment of the disclosure, the length of the peptide is 10 to 80 aa such as from 10 to 70 aa, from 10 to 60 aa or from 10 to 50 aa.

[0127] The peptides mentioned in the examples herein have a length of from 30 to 50 aa such as from 35 to 50 aa.

[0128] In one embodiment of the disclosure, the fragment has at least 50 % sequence identity with the sequence defined in SEQ ID NO 85. In one embodiment of the disclosure, the fragment has at least 50 % sequence identity with the sequence defined in SEQ ID NO 86. In one embodiment of the disclosure, the fragment has at least 50 % sequence identity with the sequence defined in SEQ ID NO 87. In one embodiment of the disclosure, the fragment has at least 50 % sequence identity with the sequence defined in SEQ ID NO 88. In one embodiment of the disclosure, the fragment has at least 50 % sequence identity with the sequence defined in SEQ ID NO 89. In one embodiment of the disclosure, the fragment has at least 50 % sequence identity with the sequence defined in SEQ ID NO 90. In one embodiment of the disclosure, the fragment has at least 50 % sequence identity with the sequence defined in SEQ ID NO 91 . In one embodiment of the disclosure, the fragment has at least 50 % sequence identity with the sequence defined in SEQ ID NO 92. In one embodiment, the sequence identity threshold is 60 %, 70 %, 80 %, 85 %, 90 %, or 95 %. In one embodiment, the polypeptide does not differ from the reference sequence in the haem binding site, whereas variation may be present relative to the reference sequence outside of the haem binding site (Meining and Skerra 2012). In one embodiment, the polypeptide does not differ from the reference sequence over the sequence GSTCPWLKKIX1(SEQ ID NO: 20). In one embodiment, the polypeptide does not differ from the reference sequence over the sequence GATEAEISMTSTX2WRKGVCEETSGAYEKTD (SEQ ID NO: 21 ). In one embodiment, the polypeptide contains the cysteine residue corresponding to position 34 of SEQ ID NO: 30. In one embodiment, the polypeptide contains the cysteine residue corresponding to position 72 of SEQ ID NO: 30. In one embodiment, the polypeptide differs from the reference sequence only by conservative mutations.

[0129] In one embodiment of the disclosure, the fragment is defined in SEQ ID NO 100, corresponding to position 23-91 of human wtA1 M. In one embodiment of the disclosure, the fragment is defined in SEQ ID NO 101 , corresponding to position 23-33 of human wtA1 M. In one embodiment of the disclosure, the fragment is defined in SEQ ID NO 102, corresponding to position 35-91 of human wtA1 M. In one embodiment of the disclosure, the fragment is SRI-36 defined in SEQ ID NO 103, corresponding to position 19-54 of human wtA1 M. In one embodiment of the disclosure, the fragment is ENF-46 defined in SEQ ID NO 104, corresponding to position 14-59 of human wtA1 M.

[0130] In one embodiment of the disclosure, the peptide has at least 50 % sequence identity with the sequence defined in SEQ ID NO 83. In one embodiment of the disclosure, the peptide has at least 50 % sequence identity with the sequence defined in SEQ ID NO 103. In one embodiment of the disclosure, the peptide has at least 50 % sequence identity with the sequence defined in SEQ ID NO 104. In one embodiment, the sequence identity threshold is 60 %, 70 %, 80 %, 85 %, 90 %, or 95 %. In one embodiment, the polypeptide does not differ from the reference sequence in the haem binding site, whereas variation may be present relative to the reference sequence outside of the haem binding site (Meining and Skerra 2012). In one embodiment, the polypeptide does not differ from the reference sequence over the sequence GSTCPWLKKIX1(SEQ ID NO: 20). In one embodiment, the polypeptide does not differ from the reference sequence over the sequence GATEAEISMTSTX2WRKGVCEETSGAYEKTD (SEQ ID NO: 21 ). In one embodiment, the polypeptide contains the cysteine residue corresponding to position 34 of SEQ ID NO: 30. In one embodiment, the polypeptide contains the cysteine residue corresponding to position 72 of SEQ ID NO: 30. In one embodiment, the polypeptide differs from the reference sequence only by conservative mutations.

[0131] In one embodiment of the disclosure the peptide is the sequence Y-F11 -K, where F11 has at least 50 % sequence identity with the sequence defined in SEQ ID NO 100. In one embodiment of the disclosure the peptide is the sequence Y-F12-C, where F12 has at least 50 % sequence identity with the sequence defined in SEQ ID NO 101 . In one embodiment of the disclosure the peptide has the sequence C-fl3-K, where F13 has at least 50 % sequence identity with the sequence defined in SEQ ID NO 102.

[0132] The present disclosure provides a peptide having a length of from 10 to 100 amino acid residues, wherein the peptide comprises a fragment selected from position 1 to position 183 of human wt alpha-1 -microglobulin, A1 M, SEQ ID NO: 30, wherein the fragment has a length of from 10 to 100 amino acid residues, and wherein the fragment includes one or more of Y22, C34, K69, K92, K118, H122, Y132, L180, 1181 , P182 and R183.

[0133] The fragment may be selected from position 1 to position 183 of human wtA1 M, and wherein the fragment has at least 50% identity with the corresponding fragment of human wtA1 M.

[0134] The fragment typically has a length of from 10 to 80 amino acid residues such as from 10 to 70, from 15 to 66 or from 20 to 75 amino acid residues.

[0135] The fragment selected typically includes i) one or more of Y22, C34, K69, K92, K118, H122, and Y132, ii) one or more of K92, K1 18, H122, Y132, L180, 1181 , P182 and R183, or ill) one or more of L180, 1181 , P182 and R183.

[0136] Typically, the fragment is selected i) from position 22 to position 183 of human wtA1 M, SEQ ID NO: 105, ii) from position 22 to position 160 of human wtA1 M, SEQ ID NO: 106, iii) from position 51 to position 155 of human wtA1 M, SEQ ID NO: 107, iv) from position 59 to position 135 of human wtA1 M, SEQ ID NO: 108, v) from position 92 to position 183 of human wtA1 M, SEQ ID NO: 109, or vi) from position 92 to position 135 of human wtA1 M, SEQ ID NO: 1 10.

[0137] The peptides of the disclosure have a length of at the most 100 aa (amino acids), notably at the most 80 aa such as at the most 75 aa, at the most 71 aa, at the most 60 aa, at the most 55 aa, at the most 50 aa such as at the most 49 aa, at the most 48 aa, at the most 47 aa, at the most 46 aa, at the most 45 aa, at the most 44 aa, at the most 43 AA, at the most 42 aa, at the most 41 aa, at the most 40 aa, at the most 39 aa, at the most 38 aa, at the most 37 aa, at the most 36 aa, at the most 35 aa, at the most 34 aa, at the most 33 aa, at the most 32 aa, at the most 31 aa, at the most 30 aa, at the most 29 aa, at the most 28 aa or at the most 27 aa. In general, the minimal length of the peptides of the disclosure is 10 aa. In general, the length of the peptides of the disclosure is from 10 to 80 aa such as from 10 to 70 aa, from 10 to 60 aa or from 10 to 50 aa. The peptides mentioned in the examples herein have a length of from 30 to 50 aa such as from 35 to 50 aa. A peptide of the present disclosure may have an amino acid sequence that is identical with a part of amino acid sequence of the human wild type A1 M (SEQ ID NO: 30), i.e. no changes apart from the length. Alternatively, a peptide of the present disclosure may have a sequence identity of 50% or more such as 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more with a part of the amino sequence of the human wild type A1 M, i.e. both a change in length and in specific amino acids present in the amino acid sequence. Alternatively, a peptide of the present disclosure may have a similarity of 50% or more such as 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more with a part of the amino sequence of the human wild type A1 M, i.e. both a change in length and in specific amino acids present in the amino acid sequence.

[0138] Whenever a specific amino acid sequence is mentioned herein it is understood that a peptide according to the disclosure (or, if the sequence only refers to a part of a peptide of the disclosure) may have a sequence identity of 50% or more such as 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more or 100% with the specific amino acid sequence. This applies inter alia to SEQ ID Nos: 30 and 103-116.

[0139] In specific embodiments, whenever a specific amino acid sequence is mentioned herein it is understood that a peptide according to the disclosure (or, if the sequence only refers to a part of a peptide of the disclosure) may have a similarity of 50% or more such as 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more or 100% with the specific amino acid sequence. This applies inter alia to SEQ ID NOs: 30 and 103-116.

[0140] A peptide according to the disclosure may comprise a fragment selected from one of the following ranges of human wtA1 M: i) Position 22 to position 135, SEQ ID NO: 110, ii) Position 26 to position 135, SEQ ID NO: 111 , ill) Position 30 to position 135, SEQ ID NO: 112, iv) Position 31 to position 135, SEQ ID NO: 113, v) Position 61 to position 135, SEQ ID NO: 114, vi) Position 66 to position 135, SEQ ID NO: 115, vii) Position 87 to position 135, SEQ ID NO: 116, viii) Position 92 to position 135, SEQ ID NO: 117.

[0141] A peptide according to the disclosure may comprise an amino acid sequence having a sequence identity of 50% or more of one of the following sequences of human wtA1 M: i) Position 22 to position 135, ii) Position 26 to position 135, ill) Position 30 to position 135, iv) Position 31 to position 135, v) Position 59 to position 135, vi) Position 66 to position 135, vii) Position 87 to position 135, viii) Position 92 to 135.

[0142] A peptide according to the disclosure may comprise an amino acid sequence selected from one of the following sequences of human wtA1 M: i) Position 59 to position 135, ii) Position 66 to position 135, ill) Position 87 to position 135, iv) Position 92 to position 135.

[0143] Moreover, a peptide according to the disclosure may at the N-terminal end and or the C-terminal comprises an amino acid sequence, Z, having a length of from 1 to 20 amino acid residue.

[0144] Z may comprise: iv) one or more lysine, K, residue(s), one or more glutamic acid, E, residue(s), one or more arginine, R, residue(s) and / or one or more aspartic acid, D, residue(s), v) one or more hydrophobic amino acid residue(s), vi) one or more of histidine H, residue(s), one or more of glutamine, G, residue(s), and / or one or more of isoleucine, I residue(s), and / or at the N-terminal end Z is or comprises one or more of the following sequences: v) HHHHHHHHGGGGGIEGR (8H5GIEGR) (SEQ ID NO: 93), vi) HHHHHHHHDDDDK (8H4DK) (SEQ ID NO: 94), vii) HHHHHHDDDDK (6H4DK) (SEQ ID NO: 95), or viii) HHHHHHHH (8H) (SEQ ID NO: 96), and / or at the C-terminal end Z is or comprises one or more of the following sequences: v) RGIEGGGGGHHHHHHHH (RGIE5G8H) (SEQ ID NO: 97) vi) KDDDDHHHHHHHH (K4D8H) (SEQ ID NO: 98) vii) KDDDDHHHHHH (K4D6H) (SEQ ID NO: 99), or viii) HHHHHHHH (8H) (SEQ ID NO: 96),

[0145] As it appears from the examples herein, peptides of the disclosure include i) an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 103, which is SR IYGKWYNLAI GSTCPWLKKI MDRMTVSTLV LGEG (corresponding to SRI-36, pos 19-54 of wtA1 M), ii) an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 104, which is ENFNISR IYGKWYNLAI GSTCPWLKKI MDRMTVSTLV LGEGATEAE (corresponding to ENF-46, pos 14-59 of wtA1 M)

[0146] In one embodiment of the disclosure, the peptide has a length from 10 to 100 amino acid residues wherein the peptide comprises a fragment selected from position 1 to position 183 of human wt A1 M, as defined in sequence SEQ ID 30. In one embodiment of the disclosure, the fragment has a length from 10 to 100 amino acid residues. In one embodiment of the disclosure, the fragment includes one or more of Y22, C34, K69, K92, K118, H122, Y132, L180, 1181 , P182 and R183.

[0147] In one embodiment of the disclosure, the fragment has at least 50 % sequence identity with a fragment selected from position 1 to position 183 of human wt A1 M.

[0148] In one embodiment of the disclosure, the fragment typically has a length of 10-80 amino acid residues, such as from 10 to 70, from 15 to 66 or from 20 to 75 amino acid residues.

[0149] In one embodiment the fragment typically includes one or more of Y22, C34, K69, K92, K118, H122, and Y132.

[0150] In one embodiment the fragment typically includes one or more of K92, K118, H122, Y132, L180, 1181 , P182 and R183.

[0151] In one embodiment the fragment typically includes one or more of L180, 1181 , P182 and R183. In one embodiment of the disclosure, the fragment is the sequence defined in SEQ ID NO 105, corresponding to position 22-183 of human wtA1 M. In one embodiment of the disclosure, the fragment is the sequence defined in SEQ ID NO 106, corresponding to position 22-160 of human wtA1 M. In one embodiment of the disclosure, the fragment is the sequence defined in SEQ ID NO 107, corresponding to position 51 -155 of human wtA1 M. In one embodiment of the disclosure, the fragment is the sequence defined in SEQ ID NO 108, corresponding to position 59-135 of human wtA1 M. In one embodiment of the disclosure, the fragment is the sequence defined in SEQ ID NO 109, corresponding to position 92-183 of human wtA1 M. In one embodiment of the disclosure, the fragment is the sequence defined in SEQ ID NO 110, corresponding to position 22-135 of human wtA1 M.

[0152] In one embodiment of the disclosure, the peptide has a length of at the most 100 amino acid residues, notably at the most 80 residues such as at the most 75 residues, at the most 71 residues, at the most 60 residues, at the most 55 residues, at the most 50 residues such as at the most 49 residues, at the most 48 residues, at the most 47 residues, at the most 46 residues, at the most 45 residues, at the most residues aa, at the most 43 residues, at the most residues aa, at the most 41 residues, at the most 40 residues, at the most 39 residues, at the most 38 residues, at the most 37 residues, at the most 36 residues, at the most 35 residues, at the most 34 residues, at the most 33 residues, at the most 32 residues, at the most 31 residues, at the most 30 residues, at the most 29 residues, at the most 28 residues or at the most 27 residues. In one embodiment of the disclosure, the minimal length of the peptide 10 residues. In one embodiment of the disclosure, the length of the peptide is from 10 to 80 residues, such as from 10 to 70 residues, from 10 to 60 residues or from 10 to 50 residues. The peptides mentioned in the examples herein have a length of from 30 to 50 residues such as from 35 to 50 residues.

[0153] In one embodiment of the disclosure, the fragment is defined in SEQ ID NO 111 , corresponding to position 26-135 of human wtA1 M. In one embodiment of the disclosure, the fragment is defined in SEQ ID NO 112, corresponding to position SO- 135 of human wtA1 M. In one embodiment of the disclosure, the fragment is defined in SEQ ID NO 113, corresponding to position 31-135 of human wtA1 M. In one embodiment of the disclosure, the fragment is defined in SEQ ID NO 114, corresponding to position 61-135 of human wtA1 M. In one embodiment of the disclosure, the fragment is defined in SEQ ID NO 115, corresponding to position 66- 135 of human wtA1 M. In one embodiment of the disclosure, the fragment is defined in SEQ ID NO 116, corresponding to position 87-135 of human wtA1 M. In one embodiment of the disclosure, the fragment is defined in SEQ ID NO 117, corresponding to position 92-135 of human wtA1 M. In one embodiment of the disclosure, the fragment is SRI-36 defined in SEQ ID NO 103, corresponding to position 19-54 of human wtA1 M. In one embodiment of the disclosure, the fragment is ENF-46 defined in SEQ ID NO 104, corresponding to position 14-59 of human wtA1 M.

[0154] In one embodiment of the disclosure, the peptide has at least 50 % sequence identity with the sequence defined in SEQ ID NO 30. In one embodiment of the disclosure, the peptide has at least 50 % sequence identity with the sequence defined in SEQ ID NO 105. In one embodiment of the disclosure, the peptide has at least 50 % sequence identity with the sequence defined in SEQ ID NO 106. In one embodiment of the disclosure, the peptide has at least 50 % sequence identity with the sequence defined in SEQ ID NO 107. In one embodiment of the disclosure, the peptide has at least 50 % sequence identity with the sequence defined in SEQ ID NO 108. In one embodiment of the disclosure, the peptide has at least 50 % sequence identity with the sequence defined in SEQ ID NO 109. In one embodiment of the disclosure, the peptide has at least 50 % sequence identity with the sequence defined in SEQ ID NO 110. In one embodiment of the disclosure, the peptide has at least 50 % sequence identity with the sequence defined in SEQ ID NO 111. In one embodiment of the disclosure, the peptide has at least 50 % sequence identity with the sequence defined in SEQ ID NO 112. In one embodiment of the disclosure, the peptide has at least 50 % sequence identity with the sequence defined in SEQ ID NO 113. In one embodiment of the disclosure, the peptide has at least 50 % sequence identity with the sequence defined in SEQ ID NO 114. In one embodiment of the disclosure, the peptide has at least 50 % sequence identity with the sequence defined in SEQ ID NO 115. In one embodiment of the disclosure, the peptide has at least 50 % sequence identity with the sequence defined in SEQ ID NO 116. In one embodiment of the disclosure, the peptide has at least 50 % sequence identity with the sequence defined in SEQ ID NO 117. In one embodiment of the disclosure, the peptide has at least 50 % sequence identity with the sequence defined in SEQ ID NO 103. In one embodiment of the disclosure, the peptide has at least 50 % sequence identity with the sequence defined in SEQ ID NO 104. In one embodiment, the polypeptide of the disclosure is an isolated polypeptide. It will be understood that the term "isolated" in this context means that the polypeptide has been removed from or is not associated with some or all of the other components with which it would be found in its natural state. For example, an "isolated" polypeptide may be removed from other amino acid sequences within a larger polypeptide sequence, or may be removed from natural components such as unrelated proteins. For the sake of clarity, an "isolated" polypeptide also includes a polypeptide which has not been taken from nature but rather has been prepared de novo, such as for example by chemically synthesis and / or by recombinant methods. As described herein an isolated polypeptide described herein may be included as a component part of a longer polypeptide or fusion polypeptide.

[0155] The agent of the disclosure may comprise a second polypeptide, such as a second polypeptide that is not A1 M, derived from A1 M or an A1 M variant. Thus, one embodiment provides for an agent comprising a polypeptide as disclosed herein and a second polypeptide, such as a second polypeptide that is not A1 M, derived from A1 M, or an A1 M variant. By “A1 M, derived from A1 M or an A1 M variant” is meant a polypeptide having a sequence that is A1 M, an A1 M variant having at least 60, 70, 80, or 90 % sequence identity thereto, or a fragment of A1 M, such as a fragment that is at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid residues long.

[0156] In one embodiment, the polypeptide does not have the sequence of SEQ ID NO: 137.

[0157] Modifications

[0158] The presently disclosed polypeptides may optionally comprise one or more modifications. “Modification” as used herein includes any functionalisation of the polypeptide, e.g. via formation of a covalent bond between any atom in the polypeptide and an atom in another compound, or a covalent bond between two atoms in the polypeptide itself, or to another similar to different polypeptide.

[0159] In one embodiment, the polypeptide disclosed herein comprises one or more further modifications. In one embodiment, one or more amino acid residue of the polypeptide is optionally modified. Modifications may be with other compounds that are derived from other peptides (e.g. comprising or consisting of one or more amino acids), or the modifications may be with other moieties that are not based on amino acids. In one embodiment of the present disclosure, the further modification is a non-peptide modification. In one embodiment of the present disclosure, the further modification is a peptide-based (i.e. amino-acid based) modification.

[0160] In one embodiment, the modification disclosed herein is achieved by conjugation to the disclosed polypeptide via a linking moiety.

[0161] In one embodiment, the polypeptide is provided as a TFA salt or a chloride salt.

[0162] Substitutions

[0163] In one embodiment, the further modification is a replacement of one or more atoms or of one or more moieties.

[0164] In one embodiment, the polypeptide is further modified on the N-terminal end. In one embodiment, the polypeptide is further modified on the C-terminal end. In one embodiment, the polypeptide is further modified on a side chain. In one embodiment, the polypeptide is modified on one position, such as two positions, such as three positions, such as four, five, six, seven, eight, nine, or ten positions.

[0165] Synthetically produced polypeptides, such as those synthesized on a solid support, will sometimes comprise one or more modifications compared to the parent amino acid sequence, as they are released from the support. Such modifications may be due to certain protecting groups employed to facilitate the synthesis of the polypeptide, or the modifications may be due to the specific linker used to fix the polypeptide to the solid support. For example, some linkers will release synthesised polypeptide as a C- terminal amide. In one embodiment, the polypeptide is acetylated N-terminally. In one embodiment, the polypeptide is amidated C-terminally.

[0166] Non-natural amino acids can be employed to modify the properties of a polypeptide. Thus, in one embodiment, the polypeptide comprises a substitution with a non-natural amino acid. In one embodiment, the polypeptide comprises one or more insertions of one or more non-natural amino acids into the parent amino acid sequence of the polypeptide. In one embodiment of the present disclosure, the polypeptide comprises a substitution with a D-amino acid. In one embodiment, the polypeptide comprises an insertion of a D-amino acid. In a specific embodiment of the present disclosure, a residue (e.g. an L-amino acid) in the polypeptide is replaced with the same amino acid in D-configuration. By “same amino acid in D-configuration” is meant that the same parent amino acid is considered, but that the two amino acids differ in the stereochemical configuration at the alpha carbon. By way of example, an L-arginine residue may be replaced with a D-arginine residue. In one embodiment, the polypeptide comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more substitutions with D-amino acids.

[0167] In a specific embodiment, the polypeptide comprises a sequence selected from the group consisting of: i. NLAIGSTCP[D-Trp]LKKIMDR (SEQ ID NO: 66), ii. NLAIGSTCPW[D-Leu]KKIMDR (SEQ ID NO: 67), ill. NLAIGSTCPWLK[D-Lys]IMDR (SEQ ID NO: 68), iv. GSTCPWLK[D-Lys]IMDRMTVSTLVLGEG (SEQ ID NO: 69), v. GSTCPWLKKIMD[D-Arg]MTVSTLVLGEG (SEQ ID NO: 70), vi. GSTCPWLKKIMDRMTVSTLV[D-Leu]GEG (SEQ ID NO: 71 ), vii. GSTCPWLKKIMDRMTVSTL[D-Asp]LGEG (SEQ ID NO: 72), viii. G[D-Ser]STCPWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 81 ), and ix. [D-Asn]LAIGSTCPWLKKIMDR (SEQ ID NO: 82), or a sequence having at least 70 % sequence identity thereto, such as at least 75 %, at least 80 %, at least 85 %, at least 90 %, such as at least 95 % sequence identity thereto.

[0168] In one embodiment, the polypeptide is selected from the group consisting of: i. NLAIGSTCP[D-Trp]LKKIMDR (SEQ ID NO: 66), ii. NLAIGSTCPW[D-Leu]KKIMDR (SEQ ID NO: 67), ill. NLAIGSTCPWLK[D-Lys]IMDR (SEQ ID NO: 68), iv. GSTCPWLK[D-Lys]IMDRMTVSTLVLGEG (SEQ ID NO: 69), v. GSTCPWLKKIMD[D-Arg]MTVSTLVLGEG (SEQ ID NO: 70), vi. GSTCPWLKKIMDRMTVSTLV[D-Leu]GEG (SEQ ID NO: 71 ), vii. GSTCPWLKKIMDRMTVSTL[D-Asp]LGEG (SEQ ID NO: 72), viii. G[D-Ser]STCPWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 81 ), and ix. [D-Asn]LAIGSTCPWLKKIMDR (SEQ ID NO: 82).

[0169] In one embodiment, one, two or more methionine residues have been substituted with norleucine (NLe or Nle). In a specific embodiment, the methionine residue corresponding to M41 of A1 M has been substituted with norleucine. In one embodiment, the methionine residue corresponding to M44 of A1 M has been substituted with norleucine. In one embodiment both the methionine residues corresponding to M41 and M44 of A1 M has been substituted with norleucine.

[0170] In one embodiment, the polypeptide is selected from the group consisting of: i. GSTCPWLKKI[Nle]DR[Nle]TVSTLVLGEG (SEQ ID NO:139), ii. GSTCPWLKKI[Nle]DR[Nle]TVSTL[D-Asp]LGEG (SEQ ID NO:140), ill. GSTCPWLKKI[Nle]DR[Nle]TVSTLVLGEG[D-Ala][D-Thr][D-Glu] (SEQ ID NO:141), iv. GSTCPWLKKI[Nle]DR[Nle]TKSTLVLGEG (SEQ ID NO:142), v. EEKYNLAIGSTCPWLKKI[Nle]DR[Nle]TVSTLVLGE (SEQ ID NO:143), and vi. GSTCPWLKKI[Nle]DR[Nle]TVSTL[D-Asp]LGEG (SEQ ID NO:144).

[0171] In one embodiment, the polypeptide is selected from the group consisting of: i. GSTCPWLKKIMDRMTKSTL[d-D]LGEG (SEQ ID NO:146) ii. EEKYNLAIGSTCPWLKKIMDRMTESTLDLGE (SEQ ID NO:147) ill. EEKYNLAKGSTCPWLKKIMDRMTESTLDLGE (SEQ ID NO:148) iv. GSTCPWLKKI[NLe]DR[NLe]TKSTL[d-D]LGEG (SEQ ID NO:149) v. EEKYNLAIGSTCPWLKKI[NLe]DR[NLe]TESTLDLGE (SEQ ID NO:150) vi. EEKYNLAKGSTCPWLKKI[NLe]DR[NLe]TESTLDLGE (SEQ ID NO:151 ) vii. GSTCPWLKKI[NLe]DR[NLe]TK[N-Meth S]TL[D]LGEG (SEQ ID NO:152) viii. EEKYNLAIGSTCPWLKKI[NLe]DR[NLe]TE[N-Meth S]TLDLGE (SEQ ID NO:153) ix. EEKYNLAKGSTCPWLKKI[NLe]DR[NLe]TE[N-Meth S]TLDLGE (SEQ ID NO: 154) x. Ac-EEKYNLAIGSTCPWLKKI[Nle]DR[Nle]TKSTLVLGE-NH2 (SEQ ID NO:155) xi. Ac-EEKYNLAIGSTCPWLKKI[Nle]DR[Nle]TESTLVLGE-NH2 (SEQ ID NO: 161)

[0172] PEGylation

[0173] In one embodiment of the present disclosure, the polypeptide is PEGylated. By PEGylation is meant the functionalisation with a chemical moiety having the structure -(CH2CH2O)n-, -(CH2OCH2)n-, or -(OCH2CH2)n-.

[0174] In one embodiment, the PEGylation is at the N-terminal end, the C-terminal end, and / or on a side-chain. In one embodiment, the PEGylation is at the C-terminal end.

[0175] In one embodiment, the PEGylation is with a PEG having a molecular weight of at most 60 kDa, such as at most 50 kDa, such as at most 40 kDa, such as at most 30 kDa, such as at most 20 kDa, such as at most 15 kDa, such as at most 10 kDa, such as at most 5 kDa. In one embodiment, the PEGylation is with any one of PEG1 to PEG50. In one embodiment, the PEGylation is with any one of PEG1 to PEG30. In one embodiment, the PEGylation is with any one of PEG2 to PEG20. In one embodiment, the PEGylation is with PEG2, PEG5, PEG10, or PEG20.

[0176] In one embodiment, the PEGylated polypeptide comprises a structure selected from the group consisting of: i. GSTCPWLKKIMDRMTVSTLVLGEG-PEG2-CONH2(SEQ ID NO: 34), ii. GSTCPWLKKIMDRMTVSTLDLGEG-PEG2-CONH2(SEQ ID NO: 35), ill. AC-NLAIGSTCPWLKKIMDR-CONH2-PEG2 (SEQ ID NO: 39), iv. AC-DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGAT-PEG2 (SEQ ID NO: 40), v. NLAIGSTCPWLKKIMDR-PEG5 (SEQ ID NO: 60), vi. NLAIGSTCPWLKKIMDR-PEG10 (SEQ ID NO: 61 ), vii. GSTCPWLKKIMDRMTVSTLVLGEG-PEG5 (SEQ ID NO: 62), viii. GSTCPWLKKIMDRMTVSTLVLGEG-PEG10 (SEQ ID NO: 63), ix. NLAIGSTCPWLKKIMDR-PEG20 (SEQ ID NO: 64), and x. GSTCPWLKKIMDRMTVSTLVLGEG-PEG20 (SEQ ID NO: 65), or a sequence having at least 70 % sequence identity thereto, such as at least 75 %, at least 80 %, at least 85 %, at least 90 %, such as at least 95 % sequence identity thereto.

[0177] In one embodiment of the present disclosure, the PEGylated polypeptide is selected from the group consisting of: i. GSTCPWLKKIMDRMTVSTLVLGEG-PEG2-CONH2(SEQ ID NO: 34) ii. GSTCPWLKKIMDRMTVSTLDLGEG-PEG2-CONH2 (SEQ ID NO: 35) ill. AC-NLAIGSTCPWLKKIMDR-CONH2-PEG2 (SEQ ID NO: 39) iv. AC-DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGAT-PEG2 (SEQ ID NO: 40) v. NLAIGSTCPWLKKIMDR-PEG5 (SEQ ID NO: 60) vi. NLAIGSTCPWLKKIMDR-PEG10 (SEQ ID NO: 61 ) vii. GSTCPWLKKIMDRMTVSTLVLGEG-PEG5 (SEQ ID NO: 62) viii. GSTCPWLKKIMDRMTVSTLVLGEG-PEG10 (SEQ ID NO: 63) ix. NLAIGSTCPWLKKIMDR-PEG20 (SEQ ID NO: 64) x. GSTCPWLKKIMDRMTVSTLVLGEG-PEG20 (SEQ ID NO: 65)

[0178] Albumin binding domain functionalisation

[0179] Polypeptides of the disclosure may be further modified with other polypeptides, proteins, or fragments thereof. In one embodiment, the polypeptide further comprises an albumin binding domain or a fragment of an albumin binding domain.

[0180] In one embodiment, the albumin binding domain consists of or comprises the amino acid sequence of SEQ ID NO: 118.

[0181] In one embodiment, the albumin binding domain fragment consists of 10 to 50 amino acid residues. In one embodiment, the albumin binding domain fragment consists of or comprises the amino acid sequence of SEQ ID NO: 119. In one embodiment of the present disclosure, the albumin binding domain or fragment thereof is conjugated to the N-terminal end of the polypeptide, the C-terminal end of the polypeptide, or a side chain of the peptide.

[0182] In one embodiment of the present disclosure, the albumin binding domain or fragment thereof is conjugated to the polypeptide via a linking moiety. In one embodiment, the linker is a PEG linker. In one embodiment, the linking moiety is a peptide linker. In one embodiment, the linking moiety is selected from the group consisting of: i. GGGGGSAS (SEQ ID NO: 121 ), and ii. GGGGSGGGGSGGGGSAS (SEQ ID NO: 122).

[0183] In one embodiment, the albumin domain binding fragment comprises or consists of the amino acid sequence SDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 119).

[0184] In one embodiment, the polypeptide is selected from the group consisting of: i. GSTCPWLKKIMDRMTVSTLVLGEGSDFYKRLINKAKTVEGVEALKLHIL AALP (SEQ ID NO: 36), ii. GSTCPWLKKIMDRMTVSTLVLGEGGGGGSASSDFYKRLINKAKTVEG VEALKLHILAALP (SEQ ID NO: 37), ill. GSTCPWLKKIMDRMTVSTLVLGEGGGGGSGGGGSGGGGSASSDFY KRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 38), iv. NLAIGSTCPWLKKIMDRGGGGSASSDFYKRLINKAKTVEGVEALKLHIL AALP (SEQ ID NO: 41), v. NLAIGSTCPWLKKIMDRGGGGSGGGGSGGGGSASSDFYKRLINKAKT VEG VEALKLHILAALP (SEQ ID NO: 42), vi. DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATGGGGSASSDFY KRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 43), and vii. DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATGGGGSGGGGS GGGGSASSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 44), or a sequence having at least 70 % sequence identity thereto, such as at least 75 %, at least 80 %, at least 85 %, at least 90 %, such as at least 95 % sequence identity thereto. In one embodiment, the polypeptide is selected from the group consisting of: i. GSTCPWLKKIMDRMTVSTLVLGEGSDFYKRLINKAKTVEGVEALKLHIL AALP (SEQ ID NO: 36), ii. GSTCPWLKKIMDRMTVSTLVLGEGGGGGSASSDFYKRLINKAKTVEG VEALKLHILAALP (SEQ ID NO: 37), ill. GSTCPWLKKIMDRMTVSTLVLGEGGGGGSGGGGSGGGGSASSDFY KRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 38), iv. NLAIGSTCPWLKKIMDRGGGGSASSDFYKRLINKAKTVEGVEALKLHIL AALP (SEQ ID NO: 41), v. NLAIGSTCPWLKKIMDRGGGGSGGGGSGGGGSASSDFYKRLINKAKT VEG VEALKLHILAALP (SEQ ID NO: 42), vi. DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATGGGGSASSDFY KRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 43), and vii. DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATGGGGSGGGGS GGGGSASSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 44).

[0185] Fatty acid conjugation

[0186] Polypeptides of the disclosure may be further modified with conjugation to fatty acids, for example to improve their albumin-binding capacity. In one embodiment, the polypeptide further comprises a fatty acid. In one embodiment, the polypeptide is conjugated to a fatty acid.

[0187] In one embodiment, the fatty acid is conjugated to the N-terminal end of the polypeptide, the C-terminal end of the polypeptide, or a side chain of the peptide.

[0188] In one embodiment, the fatty acid is conjugated to the polypeptide via a linking moiety. In one embodiment, the linking moiety is yGlu-2xOEG (OEG: 8-amino-3,6- dioxaoctanoic acid; yGlu: gamma-glutamic acid). In one embodiment, the fatty acid is a C20 fatty diacid moiety [C20DA], such as octadecanoic diacid. In one embodiment, the polypeptide is conjugated to yGlu-2xOEG-C20 diacid. Other fatty acids / linkers can be conjugated to peptides to improve stability, see Zhao etal., 2022; Xu etal., 2023.

[0189] In one embodiment, the polypeptide is selected from the group consisting of: i. Ac-EEKYNLAIGSTCPWLKKI[Nle]DR[Nle]T[K([C20DA]-[yGlu]-[QEG]- [OEG]-)]STLVLGE-NH2 (SEQ ID NO:156 & SEQ ID NO:164) ii. [C20DA]-[yGlu]-[OEG]-[OEG]-

[0190] EEKYNLAIGSTCPWLKKI[Nle]DR[Nle]TKSTLVLGE-NH2 (SEQ ID NO:157) ill. Ac-GSTCPWLK[K([C20DA]-[yGlu]-[OEG]-[OEG]- )]l[NLe]DR[NLe]TK[NMeSer]TL[D]LGEG-NH2 (SEQ ID NO:158 & SEQ ID NO:165) iv. [C20DA]-[yGlu]-[QEG]-[QEG]-

[0191] GSTCPWLKKI[NLe]DR[NLe]TK[NMeSer]TL[D]LGEG-NH2 (SEQ ID NO:159)

[0192] Amino acid modifications

[0193] The polypeptides disclosed herein may comprise one or more modifications of one or more amino acid residues within the polypeptide. In one embodiment, the polypeptide comprises a modification of one or more amino acid residues.

[0194] In one embodiment, the modification is selected from the group consisting of methylation such as N-methylation, dimethylation such as N-dimethylation or C- dimethylation, side-chain methylation, oxidation such as oxidation of cysteine or methionine, and dimerization, such as dimerization between two cysteine moieties.

[0195] In one embodiment, the modification is cysteine carbamidomethylation (cysteine CAM). In one embodiment, the polypeptide comprises or consists of the amino acid sequence GSTC(Cam)PWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 48), wherein Cam specifies carbamidomethylation.

[0196] In one embodiment, the polypeptide comprises homocysteine. In one embodiment, a cysteine moiety of the polypeptide is substituted (replaced) with homocysteine. In one embodiment, the polypeptide comprises or consists of the amino acid sequence GST{hC}PWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 49), wherein hC is homocysteine.

[0197] In one embodiment, the polypeptide comprises a penicillamine residue. In one embodiment, a cysteine moiety of the polypeptide is substituted (replaced) with penicillamine. In one embodiment, the polypeptide comprises or consists of the amino acid sequence GST{Penc}PWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 50), wherein Penc is a penicillamine residue.

[0198] In one embodiment, the polypeptide comprises one or more methionine sulfoxides. In one embodiment, the polypeptide comprises or consists of the amino acid sequence GSTCPWLKKIM(ox)DRM(ox)TVSTLVLGEG (SEQ ID NO: 51 ), wherein M(ox) specifies methionine sulfoxide.

[0199] The polypeptide of the present disclosure can be alkylated, such as methylated. In one embodiment of the present disclosure, the polypeptide is methylated or dimethylated.

[0200] In one embodiment, the polypeptide comprises one or more of a modification selected from the group consisting of: i. arginine side chain methylation, ii. lysine methylation, ill. lysine dimethylation, iv. threonine N-methylation, and v. isoleucine N-methylation.

[0201] In one embodiment of the present disclosure, the polypeptide comprises or consists of an amino acid sequence selected from the group consisting of: i. GSTCPWLKKIMD[Arg methylated side chain]MTVSTLVLGEG (SEQ ID NO: 73), ii. GSTCPWLK[Lys di-methylated]IMDRMTVSTLVLGEG (SEQ ID NO: 74), ill. GSTCPWLKKIMDRM[Thr N-Methylated]VSTLVLGEG (SEQ ID NO: 75), iv. GSTCPWLKK[lle N-methylated]MDRMTVSTLVLGEG (SEQ ID NO: 76), v. NLAIGSTCPWLK[Lys di-methylated]IMDR (SEQ ID NO: 77), vi. NLAIGS[Thr N-Methylated]CPWLKKIMDR (SEQ ID NO: 78), vii. NLA[lle N-methylated]GSTCPWLKKIMDR (SEQ ID NO: 79), and viii. NLAIGSTCPWLKKIMD[Arg methylated side chain] (SEQ ID NO: 80), or a sequence having at least 70 % sequence identity thereto, such as at least 75 %, at least 80 %, at least 85 %, at least 90 %, such as at least 95 % sequence identity thereto.

[0202] Cyclised polypeptides

[0203] The polypeptides of the present disclosure may be cyclised. Cyclisation can be achieved by any known technique, i.e. a chemical modification of the polypeptide. Cyclisation can be effected for instance by reaction with a compound linking two moieties of the polypeptide. Cyclisation can also be achieved by reaction with a compound effecting reaction between two moieties on the polypeptide. In one embodiment, the polypeptide is cyclised. In one embodiment, the polypeptide comprises a linker moiety linking two amino acid residues of the polypeptide.

[0204] In one embodiment, the linking moiety has the structure of formula (I): formula (I), wherein R may be 1 , 2, 3, or 4 substituents independently selected from the group consisting of H, F, Cl, Br, I, NO2, NO, SH, SOH, SO2H, SO3H, alkyl, alkenyl, alkynyl, COOH, COOR2, CHO, OH, R2, and OR2, wherein R2is alkyl, alkenyl, or alkynyl, wherein the dashed lines specify attachment points to the amino acid residues of the polypeptide.

[0205] In one embodiment, the linking moiety has the structure of formula (la): formula (la), wherein the dashed lines specify attachment points to the amino acid residues of the polypeptide.

[0206] In one embodiment, the polypeptide comprises a linker moiety linking two amino acid residues was prepared using 1 ,3-bis(bromomethyl)benzene. In one embodiment, the two linked amino acid residues are linked via groups independently selected from an N-terminal amine, a side chain heteroatom selected from S, N, and O, a C-terminal acid, and a C-terminal amide.

[0207] In one embodiment, the polypeptide comprises or consists of a structure selected from the group consisting of: i. C1 -GSTCPWLKKIMDR-C1 -TVSTLVLGEG (SEQ ID NO: 54), ii. C1 -NLAIGSTCPWLKKIMDRMTVS-C1 (SEQ ID NO: 55), ill. C1 -AIGSTCPWLKKIMDRMT-C1 (SEQ ID NO: 56), and iv. C1 -NLAIGSTCPWLKKIMDR-C1 (SEQ ID NO: 59), or a sequence having at least 70 % sequence identity thereto, such as at least 75 %, at least 80 %, at least 85 %, at least 90 %, such as at least 95 % sequence identity thereto, wherein C1 specifies the point of attachment of the linker moiety.

[0208] In one embodiment, the polypeptide is selected from the group consisting of: i. 01 -GSTCPWLKKIMDR-C1 -TVSTLVLGEG (SEQ ID NO: 54), ii. 01 -NLAIGSTCPWLKKIMDRMTVS-C1 (SEQ ID NO: 55), ill. 01 -AIGSTCPWLKKIMDRMT-C1 (SEQ ID NO: 56), and iv. C1 -NLAIGSTCPWLKKIMDR-C1 (SEQ ID NO: 59), wherein C1 specifies the point of attachment of the linker moiety.

[0209] In one embodiment, the polypeptide is cyclised N-terminal end to C-terminal end, such as via a peptide bond. In one embodiment, the polypeptide comprises or consists of the amino acid sequence EEKYNLAIGSTCPWLKKI[Nle]DR[Nle]TKSTLVLGE (GTI-125, SEQ ID NO: 160) cyclised N-terminal end to C-terminal end.

[0210] Properties of the aoent of the disclosure

[0211] The agent of the present disclosure show A1 M activity. A1 M activity may be assessed by a number of different assays. In particular, it is recognised that A1 M activity may be assessed using the cytochrome c reduction assay. Accordingly, in one embodiment of the present disclosure, the agent is effective in reducing cytochrome c. The cytochrome c reduction activity may feasibly be compared to that of A1 M (SEQ ID NO: 30) or a functional variant thereof, such as for example the A1 M variant RMC-035 (SEQ ID NO: 16). In one embodiment of the present disclosure, the agent has at least 35 % of the cytochrome c reduction activity of SEQ ID NO: 16, such as at least 36 %, 37 %, 38 %, 39 %, 40 %, 41 %, or 42 % of the cytochrome c reduction activity of SEQ ID NO: 16.

[0212] The cytochrome c reduction assay disclosed in the examples below may be used to assess the EC50 value of a compound exhibiting A1 M activity, such as the agents of the present disclosure. In one embodiment of the present disclosure, the agent has an EC50 value for reduction of cytochrome c of at most 500 pM, such as at most 400 pM, 300 pM, 200 pM, 100 pM, or 50 pM.

[0213] The therapeutic effect of A1 M is in part due to its ability to bind heme. Free heme is cytotoxic. Accordingly, it is an advantage that A1 M binds heme, as it reduced the cytotoxic side effects. In one embodiment of the present disclosure, the agent of the present disclosure is capable of binding heme.

[0214] A1 M is an antioxidant and is therefore capable of reducing oxidative stress. In one embodiment of the present disclosure, the agent of the disclosure is an antioxidant.

[0215] Due to the similar activity of A1 M and the agents disclosed herein - as outlined in the examples below - it is contemplated that the agents of the present disclosure are effective for treatment or prophylaxis of the same diseases and disorders that A1 M and variants thereof are effective a treating or preventing.

[0216] In one embodiment of the present disclosure, the agent is capable of preventing cell death. In one embodiment of the present disclosure, the agent is capable of preventing lysis of red blood cells (RBCs).

[0217] Aqents

[0218] In one embodiment of the disclosure, the agent further comprises a moiety. In a further embodiment, this moiety is capable of altering the biophysical properties of the polypeptide. One embodiment of the present disclosure provides for an agent comprising a moiety and a biologically active polypeptide consisting of a. 12 to 80 amino acid residues, wherein the polypeptide comprises the amino acid sequence GSTCPWLKKIX1(SEQ ID NO: 20) wherein X1is M, K, R, or Nle, or b. 30 to 80 amino acid residues, wherein the polypeptide comprises the amino acid sequence GATEAEISMTSTX2WRKGVCEETSGAYEKTD (SEQ ID NO: 21 ) wherein X2is H, R, or K.

[0219] One embodiment of the disclosure provides for an agent comprising a moiety and a biologically active polypeptide consisting of 10 to 80 amino acid residues, wherein the polypeptide comprises a fragment of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 28, or SEQ ID NO: 29, wherein:

[0220] X3is selected from D, N, or G;

[0221] X4is selected from M, K, R, or Nle;

[0222] X5is selected from H, R, or K;

[0223] X6is selected from D, N, or E; and

[0224] X7is selected from M and Nle; said fragment being at least 10 amino acid residues in length, and wherein the fragment comprises an amino acid residue corresponding to C34 or C72 of SEQ ID NO: 30.

[0225] In one embodiment of the disclosure, the moiety is attached to the N-terminal of the polypeptide. In one embodiment, the moiety is attached to the C-terminal of the polypeptide. In one embodiment, the moiety is attached to a side chain of the polypeptide. The additional moiety may be a second polypeptide, a protein, or another compound. If the moiety is a second polypeptide connected to the N-terminal or C- terminal of the polypeptide of the disclosure, the moiety and the polypeptide taken together is not A1 M or a variant thereof. Specifically, in the case that the moiety is a polypeptide, the agent is an amino acid sequence having at most 95 % sequence identity to human A1 M (SEQ ID NO: 30), such as at most 90 %, 85 %, 80 %, 75 %, 70 %, 65 %, 60 %, 55 %, such as at most 50 % sequence identity to SEQ ID NO: 30. This sequence identity is calculated based on the length of SEQ ID NO: 30, i.e., it is not calculated based on the length of the shorter of the two sequences. In one embodiment, a reference to an agent comprising a polypeptide consisting of a number of amino acid residues, said polypeptide comprising a specific amino acid sequence is to also be construed as an agent comprising a polypeptide, the polypeptide comprising a specified sequence and consisting of a number of amino acid residues, wherein the N-terminal residue is not linked N-terminally to an amino acid and wherein the C-terminal residue is not linked C-terminally to an amino acid residue. In a specific embodiment, the N-terminal residue may be linked N-terminally to an amino acid residue that does not form part of the contiguous sequence of A1 M (e.g. SEQ ID NO: 30). In a specific embodiment, the C-terminal residue may be linked C- terminally to an amino acid residue that does not form part of the contiguous sequence of A1 M (e.g. SEQ ID NO: 30).

[0226] In one embodiment, a reference to an agent comprising a polypeptide consisting of a number of amino acid residues, said polypeptide comprising a specific amino acid sequence is to also be construed as an agent consisting of a polypeptide comprising a specified sequence and one or more excipients, wherein one or more amino acid residues is optionally modified.

[0227] Fusion proteins

[0228] It is contemplated that the polypeptides disclosed herein can be fused to other molecules such as other polypeptides to form the disclosed agents, without eliminating the therapeutic activity of the agents. Accordingly, one embodiment of the present disclosure provides for agent comprising the polypeptide of the disclosure and a second polypeptide. In one embodiment, the polypeptide of the disclosure is fused to albumin binding domain (SEQ ID NO: 118) or a fragment of albumin binding domain (e.g. SEQ ID NOs: 119).

[0229] In one embodiment, the second polypeptide is not a fragment of A1 M (SEQ ID NO: 120). In one embodiment, the agent comprises at most 80 contiguous amino acid residues of human A1 M (SEQ ID NO: 120) or at most 80 contiguous amino acid residues of a sequence having at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, or at least 95 % sequence identity to human A1 M (SEQ ID NO: 120). By this is meant that the agent may be longer than 80 amino acid residues, but that the part of the agent that corresponds to the A1 M fragment, or has the stated sequence identity thereto, is 80 amino acid residues or shorter. In one embodiment, the second polypeptide is albumin binding domain (ABD1 , SEQ ID NO: 118). In one embodiment, the second polypeptide is albumin binding domain fragment (SEQ ID NO: 119). In one embodiment, the second polypeptide is a Human Fc fragment, such as a Human IgG Fc fragment, such as a Human IgG 1 Fc fragment, such as a Human lgG1 Fc fragment having SEQ ID NO: 138.

[0230] Polynucleotides, vectors, and cells.

[0231] One embodiment of the present disclosure provides for a polynucleotide encoding the agent or the polypeptide disclosed herein.

[0232] One embodiment of the present disclosure provides for a vector comprising the polynucleotide disclosed herein.

[0233] One embodiment of the present disclosure provides for a cell comprising the polynucleotide disclosed herein or the vector disclosed herein.

[0234] Pharmaceutical compositions

[0235] One embodiment of the present disclosure provides for a pharmaceutical composition comprising the agent of the disclosure and a pharmaceutically acceptable carrier, excipient, or diluent. In one embodiment of the disclosure, the pharmaceutical composition is formulated for oral administration. In one embodiment, the pharmaceutical composition is formulated for subcutaneous administration, such as subcutaneous injection. In one embodiment, the pharmaceutical composition is formulated for topical administration.

[0236] One embodiment provides for an agent of the disclosure for use in medicine. One embodiment provides for an agent of the disclosure for use in the treatment of a disease or disorder.

[0237] One embodiment of the present disclosure provides for a method of treating a disease or disorder comprising administering a therapeutically effective amount of the agent of the disclosure. One embodiment of the disclosure provides for a use of the agent of the disclosure for the manufacture of a medicament for treatment of a disease or disorder.

[0238] Oxidative stress has been reported in kidney disease, and is considered an important driver of kidney pathophysiology for AKI, AKD and CKD. Accordingly, one embodiment provides for an agent of the disclosure for use in the treatment of a kidney disease, disorder or injury. In one embodiment of the disclosure, the kidney disease, disorder or injury is induced and / or propagated by or associated with oxidative stress.

[0239] One embodiment provides for a method of treatment of a kidney disease, disorder or injury comprising administration of an agent of the disclosure to a subject in need thereof.

[0240] One embodiment provides for a pharmaceutical composition for use in the treatment of a kidney disease, disorder or injury, said composition comprising the agent of the present disclosure.

[0241] One embodiment provides for a use of the agent of the present disclosure of treating a kidney condition, disorder or injury.

[0242] One embodiment provides for use of an agent of the present disclosure, characterized in that it is for the manufacture of a medicament for treatment of a kidney condition, disorder or injury.

[0243] Heme is an essential prosthetic group in an array of proteins that comprehensively affect cellular function and metabolism. However, heme and heme-related products can be damaging to the kidney due their bioreactivity and pro-oxidant effects. A key mechanism underlying the toxicity of heme and heme-related products is the induction and propagation of oxidative stress, which in turn drives various pathophysiological processes associated with kidney disease. Accordingly, in one embodiment of the disclosure, the oxidative stress is induced and / or propagated by or associated with heme and / or heme-related products. In a further embodiment of the disclosure, the oxidative stress is induced and / or propagated by or associated with cell-free heme. Haematuria is a fundamental symptom of kidney disease. Pathological mechanisms involved in kidney disease and / or injury which are associated with haematuria include release of heme and heme-related products by degradation of RBCs. The released heme and heme-related products are nephrotoxic and can accumulate within the various intra- and extracellular compartments of the kidney and induce and / or propagate oxidative stress, inflammation, cell death, and / or fibrosis, ultimately leading to deterioration of kidney function.

[0244] Accordingly, in a further embodiment of the disclosure, the kidney disease, disorder or injury presents with haematuria. In a specific embodiment, the disease or disorder is preeclampsia. In a further embodiment of the disclosure, the kidney disease, disorder or injury presents with glomerular haematuria. In one embodiment of the disclosure, the kidney disease, disorder or injury is caused by, induced by and / or propagated by glomerulonephritis. In a further embodiment of the disclosure, the kidney disease, disorder or injury is caused by, induced by and / or propagated by nephrotoxicity, such as by nephrotoxic medications. In a further embodiment of the disclosure, the kidney disease, disorder or injury is acute kidney injury (AKI). In a further embodiment of the disclosure, the acute kidney injury (AKI) is ischemia-reperfusion injury ( IRI) . In a further embodiment of the disclosure, the acute kidney injury (AKI) is caused by, induced by and / or propagated by cisplatin. In a further embodiment of the disclosure, the acute kidney injury (AKI) is caused by, induced by and / or propagated by administration of cisplatin to a subject in need thereof. In a further embodiment of the disclosure, the kidney disease, disorder or injury is acute kidney disease (AKD).

[0245] On embodiment of the present disclosure provides for a method of reducing oxidative stress in a cell, said method comprising contacting the cell with the agent of the disclosure. One embodiment of the present disclosure provides for a method of reducing oxidative in a subject, said method comprising administering the agent of the disclosure to said subject.

[0246] In a further embodiment of the disclosure, the kidney disease, disorder or injury is chronic kidney disease (CKD). In one embodiment of the disclosure, the CKD is associated with and / or presents with haematuria. In one embodiment of the disclosure, the CKD is selected from the group consisting of IgA Nephropathy, primary focal segmental glomerulosclerosis (FSGS), Alport syndrome, thin basement membrane disease (TBMN), C3 glomerulonephritis (C3GN), Lupus nephritis, ANCA-associated vasculitis, ANCA-associated glomerulonephritis, diabetic kidney disease (DKD), hypertensive nephrosclerosis, polycystic kidney disease, non-diabetic CKD, non- proteinuric CKD, interstitial nephritis, drug-induced CKD, CKD based on an autoimmune disease, CKD of unknown / unspecific causes, and CKD based on genetic abnormalities including but not limited to of APOL1 nephropathy. In one embodiment of the disclosure, the CKD is caused by glomerulonephritis.

[0247] In a further embodiment of the disclosure, the kidney disease, disorder or injury is classified according to the International Classification of Diseases 11 (ICD-11) code MF8Y, GB4Z, GB60 and / or GB61 .

[0248] In one embodiment, the disease or disorder is caused by or associated with haemorrhage, haemolysis, or anaemia, such as haemolytic anaemia. In one embodiment, the disease or disorder is haemolytic anaemia or a systemic haem disease. In one embodiment, the disease or disorder is sickle cell anaemia, haemolytic anaemia, diamond blackfan anaemia, malaria, sepsis. In one embodiment, the disease or disorder is a CNS disease or disorder, such as haemorrhagic stroke, or ischemic stroke. In one embodiment, the disease or disorder is a vascular disease or disorder, such as a cardiovascular disease or disorder. In one embodiment, the vascular disease or disorder is atherosclerosis, vascular injury, peripheral artery disease, ischemic heart disease, thrombotic vascular disease, heart failure, or myocardial infarction. In one embodiment, the disease or disorder is tissue or nerve injury due to haemolytic trauma or due to haemorrhage.

[0249] One embodiment provides for a method of treating cerebral intraventricular hemorrhage (IVH) in a subject comprising administering the agent of the disclosure.

[0250] Examples

[0251] Materials & Methods

[0252] The assay is modified from Allhorn et al. 2005 (Free Rad. Biol. Med 38:557-567) and adapted for use in 384-well plates. A cytochrome c assay substrate solution was prepared by mixing 150 pM cytochrome c (Sigma-Aldrich, C2506) and 150 pM NADH (from a 50 mM stock NADH solution in 0.01 M NaOH) in Dulbecco’s phosphate buffered saline (Gibco, 14190-094) just before starting the assay by addition to the 384-well plate. 22 pL of an RMC-035 reference protein solution (0.0085 to 70 pM), or peptide solutions (concentrations ranging from 0.06 to 97 pM) were added to a 384- well plate in duplicate. 33 pL of the assay substrate solution was quickly added to each well using a multichannel pipette. The 384 well plate was covered with plastic, mixed by shaking (linear, 900 rpm, 5 seconds) and incubated at room temperature for 2 hours, protected from light. Following incubation, the plate was placed into a multi-well plate spectrophotometer (SpectroMax i3x, Molecular Devices) for the last 5 minutes of the incubation time to adjust to temperature. The plate was mixed by shaking (linear, 900 rpm, 5 seconds), and the plate absorbance was measured at 550 nm. The absorbance values for each concentration were plotted against either RMC-035, GTI- 11 or GTI-12, or peptide concentration and the EC50 value was determined for each peptide using four parameter logistic. Each experimental peptide was compared to a positive control as stated in figures, which was either RMC-035 (the full length recombinant modified A1 M protein) or a peptide with previous effect in the cytochrome C reduction assay (GTI-11 or GTI-12). Efficacy was determined as a % of the maximum cytochrome C reduction activity of the positive control, or as an EC50 concentration for each peptides cytochrome C reduction activity.

[0253] All peptides as tested have N-terminal acetylation and C-terminal amidation and were synthesized as Trifluoroacetic acid (TFA) salts without removal of the residual TFA.

[0254] GTI-1 to GTI-4 and GTI-6 to GTI-14 are designed to comprise a residue corresponding to C34 and / or C72 of A1 M. GTI-5 is a negative control, being designed as a scrambled polypeptide. GTI-15 is a negative control, being designed as a fragment of A1 M which does not comprise a residue corresponding to C34 or C72.

[0255] GTI-16 to GTI-18 are designed with amino acid substitution and addition to enhance solubility.

[0256] GTI-24, GTI-25, GTI-29 and GTI-30 have polyethylene glycol (PEG2, {2-[2-(Fmoc- amino)ethoxy]ethoxy}acetic acid, C21 H23NO6) C-terminal conjugations as first attachment on resin, designed to enhance plasma / blood stability and pharmacokinetic profile.

[0257] GTI-31 has a flexible C-terminal GS linker (GGGGGSAS; SEQ ID: 121) to a modified albumin binding domain peptide sequence (SDFYKRLINKAKTVEGVEALKLHILAALP; SEQ ID: 119). GTI-36 is a chloride salt of GTI-11 . GTI- 37 is a cysteine to cysteine (S-S) dimer of GTI- 11 . GTI-38 to GTI-40 are modifications of GTI-11 where the cysteine at position 4 is modified as a iodoacetamide, homocysteine or penacillamide-cysteine respectively. GTI-41 is a variant of GTI-11 with methionine amino acids at positions 11 and 14 are substituted with oxidized methionine amino acids.

[0258] GTI-42 and GTI-43 are shortened GTI-11 sequences with 4 and 10 amino acids respectively.

[0259] GTI-44 to GTI-46 and GTI-50 are constrained using Chemical Linkage of Peptides onto Scaffolds (CLIPS) technology to enhance plasma / blood stability with the CLIPS scaffold attached to differing amino acids in the peptide sequence.

[0260] GTI-58 to 64 contain D-amino acid substitutions designed to enhance plasma / blood stability.

[0261] GTI-65 to GTI-72 contain side chain methylation and N-methylation variants to enhance peptide plasma residence time.

[0262] All peptides as tested have N-terminal acetylation and C-terminal amidation and were synthesized as Trifluoroacetic acid (TFA) salts without removal of the residual TFA.

[0263] GTI-73 is the GTI-11 sequence containing a D-amino acid at position 2 (serine) G[S]TCPWLKKIMDRMTVSTLVLGEG

[0264] A full-length albumin binding domain (ABD) to the peptides conjugated with a linker in order to improve systemic residence time without compromising on potency, the ABD is LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP (Hereafter termed ABD1 ; SEQ ID NO: 118). A linker is used to conjugate ABD1 to the peptides (GGGGGSAS; SEQ ID NO: 121) hereafter termed GSlink.

[0265] GTI-75 is GTI-11 base sequence with ABD1 sequence on the N-terminal with a GSlink (SEQ ID: 131 , ABD1-GSIink-GSTCPWLKKIMDRMTVSTLVLGEG), GTI-76 is GTI-11 base sequence with ABD1 sequence on the C-terminal with a GS-link (SEQ ID NO: 132, GSTCPWLKKIMDRMTVSTLVLGEG-GSLink-ABD1 ).

[0266] GTI-87 and GTI-90 peptides have also been designed with ABD1 and GSlink. These also have variants with repeated GSlink and GTI-82 to GTI-86 sequences.

[0267] • GTI-87: ABD1 -GSIink-GTI-82 x1 (SEQ ID NO: 133). GTI-88: ABD-1 -(GS-link-GTI-82 x3) (SEQ ID NO: 134).

[0268] GTI-89: ABD1 -GSIink-GTI-86 x1 (SEQ ID NO: 135).

[0269] GTI-90: ABD-1 -(GS-link-GTI-86 x3) (SEQ ID NO: 136).

[0270] Metabolism studies with peptides incubated hepatocytes and plasma indicate a potential site of metabolism at position 15, threonine. Therefore, peptides are designed with modifications to improve stability in that area as well as to improve solubility.

[0271] GTI-77 to 79 are GTI-1 1 base sequence with the either the threonine at position 15, the valine at position 16, or both the threonine and valine at positions 16 and 16 substituted for a D-amino acids.

[0272] • GTI-77: GSTCPWLKKIMDRM[T]VSTLVLGEG (SEQ ID NO: 123).

[0273] • GTI-78: GSTCPWLKKIMDRMT[V]STLVLGEG (SEQ ID NO: 124).

[0274] • GTI-79: GSTCPWLKKIMDRM[T][V]STLVLGEG (SEQ ID NO: 125).

[0275] GTI-82 and GTI-83 are GTI-1 1 sequences with the valine at position 16 replaced with either a glutamic acid or a lysine.

[0276] • GTI-82: GSTCPWLKKIMDRMT[E]STLVLGEG (SEQ ID NO: 126).

[0277] • GTI-83: GSTCPWLKKIMDRMT[K]STLVLGEG (SEQ ID NO: 127).

[0278] GTI-2 is a potent peptide that contains a DDDDK enterokinase cleavage site sequence. This have been removed and replaced with solubilizing glutamic acids as well as removing a hydrophobic C-terminal region:

[0279] • GTI-84 EEEEKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGAT (SEQ ID NO: 128).

[0280] • GTI-85 EEKYNLAIGSTCPWLKKIMDRMTVSTLVLGE (SEQ ID NO: 129).

[0281] • GTI-86 EEKYNLAIGSTCPWLKKIMDRMT[E]STLVLGE (valine position 24 substituted with Glutamic acid for solubility) (SEQ ID NO: 130).

[0282] Results

[0283] The absorbance values and the corresponding curve fits of GTI-1 to GTI-15 are shown in Figure 1 . EC50 values for polypeptides GTI-1 to GTI-18, as assessed by the cytochrome c reduction assay, are shown in the Table 1 . Figure 2 shows a summary of the cytochrome c reduction efficacy of polypeptides GTI-1 to GTI-15. Figure 3 shows a summary of the EC50 values of polypeptides GTI-1 to GTI-15. RMC-035 reduces oxidized cytochrome C with an EC50 of 0.8 pM. Polypeptides with retained A1 M reductase activity demonstrated EC50 values in the low pM range. Polypeptides GTI-1 to GTI-4, GTI-5 to GTI-14, and GTI-16 to GTI-18 show greater than 30 % of the efficacy of RMC-035. Negative controls; GTI-5 and GTI-15 provided low efficacies whereby they did not show greater than 30% of the efficacy of RMC-035, and they did not provide reduction profiles that could be assigned an EC50 values, specifically, because the obtained curves were not sigmoidal. The fitting of the GTI-5 and GTI-15 data also provided low R squared values.

[0284] Table 1 : Efficacy compared to RMC-035 (%) and EC50 values for polypeptides GTI-1 to GTI-18 as assessed by the cytochrome c reduction assay. Efficacy compared to GTI-11 (%) and EC50 values for polypeptides GTI-24 to GTI-50, as assessed by the cytochrome c reduction assay, are shown in the Table 2.

[0285] Polypeptides GTI-24 to GTI-36, GTI-39 to GTI-41 , and GTI-44 to GTI-74 show greater than 71 % of the efficacy of GTI-11 . GTI-37, GTI-38 and GTI-42 show greater than 14% of the efficacy of GTI-11 .

[0286] Table 2: Efficacy compared to GTI-11 (%) and EC50 values for polypeptides GTI-

[0287] 24 to GTI-50 as assessed by the cytochrome c reduction assay.

[0288] Efficacy compared to GTI-12 (%) and EC50 values for polypeptides GTI-54, GTI- 57, GTI-75, GTI-77 to GTI-79, GTI-82 to GTI-90, as assessed by the cytochrome c reduction assay, are shown in the Table 3. Polypeptides GTI-54, GTI- 57, GTI-75, GTI- 77 to GTI-79, GTI-82 to GTI-90 show at least 41 % greater efficacy of GT I - 12.

[0289] Table 3: Efficacy compared to GTI-12 (%) and EC50 values for polypeptides GTI- 54, GTI- 57, GTI-75, GTI-77 to GTI-79, GTI-82 to GTI-86, and GTI-89 as assessed by the cytochrome c reduction assay

[0290] Table 4: Efficacy compared to GTI-86 (%) and EC50 values for polypeptides GTI- 111 to GTI-119 as assessed by the cytochrome c reduction assay.

[0291] Efficacy compared to GTI-11 (%) and EC50 values for polypeptides GTI-111 to GTI-

[0292] 119, as assessed by the cytochrome c reduction assay, are shown in the Table 4.

[0293] Polypeptides GTI-111 , GTI-114, GTI-116, GTI-117, GTI-118, and GTI-119 show at least comparable efficacy to that of GTI-86.

[0294] Conclusion

[0295] RMC-035 reduces oxidized cytochrome C with an EC50 of 0.8 pM. Many polypeptides have reducing activity which is close to the RMC-035 potency.

[0296] The polypeptides GTI-1 to GTI-4, GTI-5 to GTI-14, and GTI-16 to GTI-18 were all capable of reducing cytochrome c in a dose dependent manner, said polypeptides having at least 30 % of the full activity of positive control RMC-035. The negative control sequences, scrambled sequences without cysteine amino acids (GTI-5 and GTI-15), showed no activity compared to RMC-035. Peptides modified with PEG2 conjugations (GTI-24, 25, 29, and 30) and added albumin binding domain sequence with GS linker (GTI-31 , GTI-75, GTI-89) retained dose dependent cytochrome C reducing potential with at least 41% of GTI-11 or GTI-12 activity. Modifications to GTI-11 and GTI-2 to introduce more soluble residues (GTI-82 to GTI-86) also maintained or improved reducing activity in cytochrome C reduction assays.

[0297] The addition of D-amino acid substitutions or methylated amino acids (GTI-58 to GTI- 74, and GTI-77 to GTI-79) did not negatively impact peptide reducing activity in Cytochrome C reduction assay.

[0298] Scrambled amino acid sequences containing a cysteine showed cytochrome c reducing activity. GTI-37 (cysteine to cysteine SS-dimer), GTI-38 (iodoacetamide cysteine), and 4 or 10 amino acid peptides (GTI-42 & 43) did not retain cytochrome c reducing activity relative to GTI-11 .

[0299] Hybrid sequences of GTI-64 and GTI-83 (GTI-111) did not compromise peptide reducing activity in the cytochrome c reduction assay. Hybrid sequences of GTI-64 and GTI-86 with the addition of substitution of Methionine for Norleucine and N-Methyl Serine (GTI-117 and GTI-119) to introduce improved stability from methionine oxidation, retained dose dependent cytochrome C reducing potential with 100% of GTI- 86 activity.

[0300] Head-to-tail cyclization of peptide GTI-120 with the addition of a single c-terminal cysteine to facilitate cyclization (GTI-125) resulted in a maintained reducing potency, EC50 or 3.35 for GTI-120 compared to an EC50 of 2.21 for GTI-125.

[0301] Fatty acid conjugations (GTI-121 to GTI-124), side chain or N-terminal yGlu-2xOEG- C20 diacid (octadecanoic diacid conjugation, resulted in a maintenance of reducing potency compared to non-conjugated peptide variants.

[0302] Materials & Methods

[0303] The assay is modified from Kristiansson et al. 2020 (Free Rad. Biol. Med, “Human radical scavenger a 1 -microglobulin protects against hemolysis in vitro and a 1- microglobulin knockout mice exhibit a macrocytic anemia phenotype”) and adapted for use in 96-well plates. Blood was harvested from Sprague Dawley rats in K2EDTA- coated vacutainers. Red blood cells (RBCs) were isolated by centrifugation at 800g for 10 minutes to fractionate the blood, with the resulting RBCs washed five times by reconstituting the RBCs in phosphate buffered saline pH 7.4 and centrifugation at 800g for 10 minutes. Finally, the RBCs were reconstituted and diluted to a 4% v / v stock concentration. Heme (Hemin [Ferriprotoporphyrin IX chloride], SigmaAldrich 51280) was used to induce RBC lysis by preparing a working 0.2 mM Hemin stock solution in PBS pH 7.4. In a 96-well plate a 250 pL mixture containing: 148.5 pL PBS; 62.5 pL RBCs (1% v / v final concentration); 25 pL Hemin (20 pM final concentration); and either 14 pL of RMC-035 reference protein solution (dilutions ranging from 0.273 to 35 pM) or peptide solutions (concentrations ranging from 0.06 to 97 pM) was prepared. The plates were incubated with agitation (linear shaker 300 rpm) for 3 hours at room temperature. Following incubation, the plates were centrifuged at 500g for 5 minutes and the resulting supernatants collected. Cell lysis was determined by measuring free haemoglobin by absorbance of 100 pL supernatant at 410 nm in a multi-well plate spectrophotometer (SpectroMax i3x, Molecular Devices). In addition, lactate dehydrogenase (LDH) release was determined using a CytoTox96 Nonradioactive cytotoxicity assay kit (Promega) adapted for 384 well plates by adding 12.5 pL assay supernatant to 12.5 pL CytoTox96 assay reagent, covered with foil to protect from light and incubated for 30 minutes at room temperature. Following incubation, 12.5 pL stop solution was added to each well and the LDH determined by measuring absorbance at 490 nm in a multi-well plate spectrophotometer (SpectroMax i3x, Molecular Devices). The absorbances for LDH and free haemoglobin measurements for each concentration were plotted against RMC-035 or peptide concentration and an EC50 determined in both pM and pg / mL.

[0304] GTI-1 to GTI-4 and GTI-6 to GTI-14 are designed to comprise a residue corresponding to C34 and / or C72 of A1 M. GTI-5 is a negative control, being designed as a scrambled polypeptide. GTI-15 is a negative control, being designed as a fragment of A1 M which does not comprise a residue corresponding to C34 or C72.

[0305] GTI-16 to GTI-18 are designed with amino acid substitution and addition to enhance solubility.

[0306] GTI-24, GTI-25, GTI-29 and GTI-30 have polyethylene glycol (PEG2, {2-[2-(Fmoc- amino)ethoxy]ethoxy}acetic acid, C21 H23NO6) C-terminal conjugations as first attachment on resin, designed to enhance plasma / blood stability and pharmacokinetic profile. GTI-31 has a flexible C-terminal GS linker (GGGGGSAS; SEQ ID NO: 121) to an albumin binding domain peptide sequence (SDFYKRLINKAKTVEGVEALKLHILAALP; SEQ ID NO: 119).

[0307] GTI-36 is a chloride salt of GTI-11 . GTI- 37 is a cysteine to cysteine (S-S) dimer of GTI- 11 . GTI-38 to GTI-40 are modifications of GTI-11 where the cysteine at position 4 is modified as a iodoacetamide, homocysteine or penacillamide-cysteine respectively.

[0308] GTI-41 is a variant of GTI-11 with methionine amino acids at positions 11 and 14 are substituted with oxidized methionine amino acids.

[0309] GTI-42 and GTI-43 are shortened GTI-11 sequences with 4 and 10 amino acids respectively.

[0310] GTI-44 to GTI-46 and GTI-50 are constrained using Chemical Linkage of Peptides onto Scaffolds (CLIPS) technology to enhance plasma / blood stability with the CLIPS scaffold attached to differing amino acids in the peptide sequence.

[0311] GTI-58 to 64 contain D-amino acid substitutions designed to enhance plasma / blood stability.

[0312] GTI-65 to GTI-72 contain side chain methylation and N-methylation variants to enhance peptide plasma residence time.

[0313] GTI-73 is the GTI-11 sequence containing a D-amino acid at position 2 (serine) G[S]TCPWLKKIMDRMTVSTLVLGEG

[0314] A full-length albumin binding domain (ABD) to the peptides conjugated with a linker in order to improve systemic residence time without compromising on potency, the ABD is LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP (ABD1 ; SEQ ID NO: 118). A linker is used to conjugate ABD1 to the peptides (GGGGGSAS; GSlink; SEQ ID NO: 121).

[0315] GTI-75 is GTI-11 base sequence with ABD1 sequence on the N-terminal with a GSlink (SEQ ID: 131 , ABD1-GSIink-GSTCPWLKKIMDRMTVSTLVLGEG), GTI-76 is GTI-11 base sequence with ABD1 sequence on the C-terminal with a GS-link (SEQ ID NO: 132, GSTCPWLKKIMDRMTVSTLVLGEG-GSLink-ABD1 ).

[0316] GTI-87 and GTI-90 peptides have also been designed with ABD1 and GSlink. These also have variants with repeated GSlink and GTI-82 to GTI-86 sequences.

[0317] • GTI-87: ABD1 -GSIink-GTI-82 x1 (SEQ ID NO: 133). GTI-88: ABD-1 -(GS-link-GTI-82 x3) (SEQ ID NO: 134).

[0318] GTI-89: ABD1 -GSIink-GTI-86 x1 (SEQ ID NO: 135).

[0319] GTI-90: ABD-1 -(GS-link-GTI-86 x3) (SEQ ID NO: 136).

[0320] Metabolism studies with peptides incubated hepatocytes and plasma indicate a potential site of metabolism at position 15, threonine. Therefore, peptides are designed with modifications to improve stability in that area as well as to improve solubility.

[0321] GTI-77 to 79 are GTI-1 1 base sequence with the either the threonine at position 15, the valine at position 16, or both the threonine and valine at positions 16 and 16 substituted for a D-amino acids.

[0322] • GTI-77: GSTCPWLKKIMDRM[T]VSTLVLGEG (SEQ ID NO: 123).

[0323] • GTI-78: GSTCPWLKKIMDRMT[V]STLVLGEG (SEQ ID NO: 124).

[0324] • GTI-79: GSTCPWLKKIMDRM[T][V]STLVLGEG (SEQ ID NO: 125).

[0325] GTI-82 and GTI-83 are GTI-1 1 sequences with the valine at position 16 replaced with either a glutamic acid or a lysine.

[0326] • GTI-82: GSTCPWLKKIMDRMT[E]STLVLGEG (SEQ ID NO: 126).

[0327] • GTI-83: GSTCPWLKKIMDRMT[K]STLVLGEG (SEQ ID NO: 127).

[0328] GTI-2 is a potent peptide that contains a DDDDK enterokinase cleavage site sequence. This have been removed and replaced with solubilizing glutamic acids as well as removing a hydrophobic C-terminal region:

[0329] • GTI-84 EEEEKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGAT (SEQ ID NO: 128).

[0330] • GTI-85 EEKYNLAIGSTCPWLKKIMDRMTVSTLVLGE (SEQ ID NO: 129).

[0331] • GTI-86 EEKYNLAIGSTCPWLKKIMDRMT[E]STLVLGE (valine position 24 substituted with Glutamic acid for solubility) (SEQ ID NO: 130).

[0332] Results

[0333] LDH absorbance values for polypeptides GTI-1 to GTI-15 are shown in Figure 4, and the EC50 values for polypeptides GTI-1 to GTI-90 are shown in Table 4. RMC-035, GTI- 1 to GTI-4 and GTI-6 to GTI-12 all showed a decrease in absorption as the concentration of polypeptide increased. GTI-5 and GTI-15 showed no change in absorption, even at high polypeptide concentration. The response curves of GTI-1 to GTI-4, GTI-6 to GTI-8, and GTI-10 to GTI-12 were sigmoidal, and accordingly, EC50 values could be calculated. Table 5: EC50 values from LDH assay for polypeptides GTI-1 to GTI-90.

[0334] Free haemoglobin absorbance values are shown in Figure 5, and the EC50 values for polypeptides GTI-1 to GTI-90 are shown in Table 5. RMC-035, GTI-1 to GTI-4 and GTI- 6 to GTI-14 all showed a decrease in absorption of LDH as the concentration of polypeptide increased, indicating that these polypeptides were capable of preventing RBC lysis. GTI-5 and GTI-15 showed no change in absorption, even at high polypeptide concentration. The response curves of GTI-1 to GTI-4, GTI-6 to GTI-8, and GTI-10 to GTI-12 were sigmoidal, and accordingly, EC50 values could be calculated. Table 6: EC50 values from haemoglobin release assay for polypeptides GTI-1 to GTI-90.

[0335] Conclusion

[0336] GTI-1 to GTI-4, GTI-6 to GTI-14, GTI-16 to GTI-90 all showed a decrease in absorption of LDH once a certain threshold in the concentration of polypeptide was reached, indicating that the peptides reduced or prevented RBC lysis, indicative of a heme binding effect. For GTI-1 to GTI-4, GTI-6 to GTI-8, and GTI-10 to GTI-12, EC50 values were obtained based on the data relating to LDH absorbance. The negative controls GTI-5 and GTI-15 showed no ability to prevent RBC lysis based on LDH. The polypeptides GTI-1 to GTI-4 and GTI-6 to GTI-12 reduced or prevented RBC lysis in a dose dependent manner as assessed by the presence of free haemoglobin. For GTI-1 to GTI-4, GTI-6 to GTI-8, and GTI-10 to GTI-12, EC50 values could be obtained based on plots relating to haemoglobin absorbance. The negative controls GTI-5 and GTI-15 showed no ability to prevent RBC lysis based on free haemoglobin.

[0337] Example 3: Human Kidney-2 (HK-2) proximal tubule cell line viability assay Materials & Methods

[0338] Human kidney cortex proximal tubule epithelial cells (HK-2), ATCC® CRL- 2190™ were used in a cell viability assay to assess the effect and potency of peptides to inhibit heme-induced loss of cell viability as determined by lactate dehydrogenase (LDH release). HK-2 cells were maintained in keratinocyte SFM medium (Gibco) supplemented with 0.05 mg / mL bovine pituitary extract and 5 ng / mL epidermal growth factor. HK-2 cells were seeded into 384 well cell culture plates at a density of 8,000 cells / well in 50 pL culture medium and incubated for 18 hours in a cell culture incubator.

[0339] Heme (Hemin [Ferriprotoporphyrin IX chloride], SigmaAldrich 51280) was used to induce cytotoxicity by preparing a working 1 mM Hemin stock solution in PBS pH 7.4. In the 364-well plates containing HK-2 cells, 25 pL culture medium was removed, and 20 pL each tested peptide solution in assay medium (final well concentrations ranging from 0.068 to 70 pM) was added to relevant wells of the 384 cell culture plate containing HK-2 cells. Finally to each well, 5 pL of either PBS pH 7.4 (negative control) or 20 pM Hemin was added, and incubated for 24 hours.

[0340] After 24 hours, LDH release was assessed using a CytoTox96 Non-radioactive cytotoxicity assay (Promega, G1780) as per the manufacturer’s instructions. Five pL of 10x lysis buffer provided by the LDH assay kit was added to wells designated for positive control for LDH release. From each well, 12.5 pL assay medium was transferred to a new 384-well plate, 12.5 pL of CytoTox 96 assay reagent added to each samples which was then protected from light and incubated at room temperature for 30 minutes. After the addition of 12.5 pL assay Stop Solution, the absorbance of each well was recorded at 490 nm on a SpectraMax i3x (Molecular Devices) within 1 hour of adding the Stop Solution. The absorbances for LDH measurements for each concentration were plotted against peptide concentration and an EC50 determined in both pM and pg / mL. Results

[0341] LDH absorbance values are shown in Figure 12, and the corresponding EC50 values are shown in Table 6. RMC-035, and all peptides tested except GTI-5, GTI-5, GTI-7, GTI-13, GTI-14, and GTI-15, showed a decrease in LDH absorption as the concentration of polypeptide increased. The response curves of peptides showing inhibition of heme-mediated LDH release were sigmoidal, and accordingly, EC50 values could be calculated.

[0342] Table 7: HK-2 EC50 values from LDH assay for GTI-1 to GTI-16, GTI-18, GTI-24, GTI-25, GTI-29, GTI-30, GTI-36, GTI-39, GTI-41 , GTI-45, and GTI-46.

[0343] Conclusion

[0344] EC50 values of peptides ranged from 1.79 - 16.9pM. RMC-035, and GTI-1 to GTI-6, GTI-8, GTI-9, , GTI-10-12, GTI-24-25, GTI- 29-31 , GTI 46, GTI-50, GTI-61-64 and GTI- 74 all showed a decrease in LDH absorption as the concentration of polypeptide increased, demonstrating the capacity of the polypeptides to inhibit a heme-induced reduction in cell viability.

[0345] Scrambled random sequence (GTI-5) and a non-cysteine containing sequence (GTI- 15) did not inhibit hemin-induced loss of HK-2 cell viability. Pegylation with 2 kDa PEG (GTI-29 & GTI-30), hydrophilic amino acid substitution (GTI-16) & C-terminal hydrophilic amino acid additions (GTI-18) did not negatively affect peptide protection of HK-2 cells from hemin toxicity.

[0346] Example 4: Human Kidney-2 (HK-2) proximal tubule cell line heme oxygenase mRNA expression assay

[0347] Materials & Methods

[0348] Human kidney cortex proximal tubule epithelial cells (HK-2), ATCC® CRL- 2190™ were employed as in example 3. HK-2 cells were seeded 24-well cell culture plates at a cell density of 0.2 million cells / well in 0.5 mL keratinocyte SFM culture medium (Gibco) supplemented with 0.05 mg / mL bovine pituitary extract and 5 ng / mL epidermal growth factor and were incubated in a cell culture incubator overnight to achieve 80-90% confluence.

[0349] Heme (Hemin [Ferriprotoporphyrin IX chloride], SigmaAldrich 51280) was used to induce cytotoxicity by preparing a working 1 mM Hemin stock solution in PBS pH 7.4. To the 24-well plates containing HK-2 cells, either PBS or RMC-035 (to a final concentration of 10 pM) or peptide GTI-11 (to a final concentration of 35 pM) and Hemin (final concentration of 20 pM) were added to relevant wells following by incubation for 4 hours. After incubation, the medium was removed, and the cells washed twice with Dulbecco’s PBS (Gibco). RNA was extracted from the cells as per the PureLink™ RNA Mini Kit (Life Technologies, 12183025) protocol. Cells were lysed with 350 pL PureLink RNA extraction kit lysis buffer plus 1% 2-mercaptoethanol, and the lysates frozen immediately on dry ice, and stored at -80°C until further processing. RNA was isolated according to manufacturer’s recommendations and included On- column PureLink DNase (Life Technologies 12185-010) treatment. RMA was quantified using UV absorbance at 260 nm. cDNA was prepared from mRNA using iScript Reverse Transcription Supermix for RT-qPCR (BioRad, 1708841) according to the manufacturer’s instructions using 15 ng RNA per qPCR reaction in 20 pL in a 384 well plate. The reverse transcription reaction in a thermal cycler was priming 5 min at 25°C, reverse transcription for 20 min at 46°C, and RT inactivation for 1 min at 95°C. Realtime PCR was performed according to the manufacturer’s instructions (iTaq Universal SYBR® Green Supermix, BioRad, 1725124) using the heme oxygenase-1 (HO-1) gene sequence forward (FH1 HMOX1 , Sigma Aldrich 8816057690-10 / 0, CAACAAAGTGCAAGATTCTG) and reverse (RH1_HMOX1 , Sigma Aldrich, 8816057690-10 / 1 , TGCATTCACATGGCATAAAG) primers. Each reaction contained 10 ng / pL cDNA, 5 pM forward and reverse primers and 2.5 pL 2 x concentration MasterMix in a volume of 5 pL. The thermal cycler program was Initial denaturation 95°C for 1 min, 39 cycles of 15 sec denaturation at 95°C, Annealing at 55°C for 20 sec, and 45 sec extension at 60°C.

[0350] Results

[0351] HO-1 mRNA expression, expressed as fold-change vs PBS control treatment, are shown in Figure 13. Hemin incubation for 4 hours increased HO-1 mRNA expression 300-fold vs control levels. This hemin-induced increase in HO-1 expression was completely inhibited by concomitant incubation with RMC-035 or peptide GTI-11 . Incubation of RMC-035 or GTI-11 alone did not increase HO-1 mRNA expression.

[0352] Conclusion

[0353] HO-1 expression is induced under oxidative stress. RMC-035 and peptide GTI-11 prevent hemin-induced increases in HO-1 expression, demonstrating that they are cytoprotective against oxidative stress and / or injury. ntravenous GTI-11 in rat ischemia and

[0354] Materials & Methods

[0355] An ischemia and reperfusion ( I Rl) model of acute kidney injury (I RI-AKI) in rats was employed to assess the in vivo activity of peptides (GTI-11). Male Sprague Dawley rats aged 8-10 weeks (Charles River Laboratories, Italy) were anesthetized with isoflurane and placed on a warming pad for temperature maintenance. Under sterile conditions, a midline abdominal incision was made to facilitate access to the renal pedicles. The right kidney was removed by blunt dissection and the renal artery and vein sutured off. Ischemia of the left kidney was initiated by clamping the renal artery and vein for 40 min using non-traumatic clamps (Agnthos, Lidingb, Sweden). At the conclusion of the ischemic period, the clamp was removed, and the kidney was observed to insure rapid re-establishment of blood flow. Following conclusion of ischemia and reperfusion, the incision was sutured, and the animal allowed to recover with free access to food and water.

[0356] Vehicle (10 mM Na-Phosphate pH 7.4, 0.15 M NaCI, 2 mg / mL histidine) or GTI-11 formulated in vehicle at 10 mg / mL were administered at 0.5h pre-ischemia and 4, 8, 24 and 48 hours post reperfusion by intravenous bolus injection via the tail vein at 0.5 mL / kg, providing a final dose of 5 mg / kg. Vehicle and GTI-11 . Following surgery, rats were recovered for 5 days, with blood collected via the tail vein at Baseline pre-surgery, days 1 (24h), 2 (48h), 3 (72h) and 5 (120h) post-surgery for assessment of kidney injury biomarkers. Serum creatinine (Biovision, Milpitas, CA, USA, K625) and blood urea nitrogen (BUN) (Thermo Fisher, MA, USA, EIABUN) were assessed according to each assay kit manufacturer’s instructions.

[0357] Results

[0358] Serum creatinine (mg / dL) and BUN (mg / dL) concentrations at baseline pre-surgery, days 1 (24h), 2 (48h), 3 (72h) and 5 (120h) post-surgery are presented in Figure 14. At baseline, serum creatinine (mg / dL) and BUN (mg / dL) concentrations were comparable between vehicle and GTI-11 groups. Serum creatinine (mg / dL) and BUN (mg / dL) concentrations were significantly decreased in GTI-11 vs vehicle groups on Day 1 , 2, 3 and 5 post-surgery. Conclusion

[0359] These data show that in a rat IRI model of AKI, GTI-11 significantly inhibits IRI- mediated kidney injury when administered over 48 hours intravenously, as indicated by inhibition of changes in serum creatinine and BUN, systemic indicators of renal function. of intravenous and subcutaneous dosed GTI-2 in rat ischemia and -acute ki

[0360] Materials & Methods

[0361] An ischemia and reperfusion (IRI) model of acute kidney injury (I RI-AKI) in rats was employed to assess the in vivo activity of peptide GTI-2. Male Sprague Dawley rats aged 8-10 weeks (Charles River Laboratories, Italy) were anesthetized with isoflurane and placed on a warming pad for temperature maintenance. Under sterile conditions, a midline abdominal incision was made to facilitate access to the renal pedicles. The right kidney was removed by blunt dissection and the renal artery and vein sutured off. Ischemia of the left kidney was initiated by clamping the renal artery and vein for 40 min using non-traumatic clamps (Agnthos, Lidingb, Sweden). At the conclusion of the ischemic period, the clamp was removed, and the kidney was observed to insure rapid re-establishment of blood flow. Following conclusion of ischemia and reperfusion, the incision was sutured, and the animal allowed to recover with free access to food and water.

[0362] Vehicle (sterile phosphate buffered saline) or GTI-2 formulated in vehicle at 10 mg / mL were administered at 0.5h pre-ischemia and 4, 24 and 48 hours post reperfusion by either intravenous bolus injection via the tail vein or subcutaneous injection as follows:

[0363] • IV dose: peptide stock formulation at 10 mg / mL in PBS, vehicle or peptides dosed at 0.5 mL / kg for a total dose of 5 mg / kg

[0364] • SC dose: peptide stock formulation at 15 mg / mL, vehicle or peptides dosed by SC injection of 666 pL / kg for 10 mg / kg.

[0365] Following surgery, rats recovered for 3 days, with blood collected via the tail vein at baseline pre-surgery, days 1 (24h), 2 (48h), and 3 (72h) post-surgery for assessment of kidney injury biomarkers. pCreatinine (Biovision, Milpitas, CA, USA, K625) and blood urea nitrogen (BUN) (Thermo Fisher, MA, USA, EIABUN) were assessed according to each assay kit manufacturer’s instructions. N=7-8 per group. Real-time GFR was determined at baseline (DO), day 1 (D1 , 24h) and day 3 (D3, 72h) post-IRI surgery. Real-time GFR was assessed in isoflurane anesthetized rats by means of measuring fluorescein-isothiocyanate-labelled sinistrin (FITC-sinistrin) clearance utilizing the MediBeacon transdermal detection system (MediBeacon Transdermal Mini GFR Monitor, MediBeacon, Mannheim, Germany), which was placed on the rat shaved chest. Background measurements were obtained over a period of 5 minutes prior to tail vein injection of FITC-sinistrin (5 mg / 100 g body weight in saline). Blood FITC-sinistrin concentrations were recorded transdermally over 30-45 minutes and FITC-sinistrin clearance was calculated according to the manufacturer’s instructions using software provided by MediBeacon. The FITC-sinistrin half-life was determined and the GFR calculated by MediBeacon software using the formula GFR (mL7min / 100g body weight) = rat conversion factor 31.26 / 11 / 2 FITC-sinistrin (Ellery et al. (2015). Nephrology 20:117-123).

[0366] Results

[0367] Rat pCreatinine and BUN concentrations (mg / dL) and 3-days post-uni-nephrectomy and ischemia reperfusion injury and treatment with either saline vehicle or peptide GTI- 2 by IV or SC administration is presented in Figure 15. The concentrations of pCreatinine and BUN of rats treated with GTI-2 was significantly lower compared with those treated with vehicle at both day 1 and day 3 post-surgery. This GTI-2 induced effect on pCreatinine and BUN concentrations was observed following both IV and SC administration.

[0368] Rat real-time glomerular filtration rate (GFR) 3-days post-uni-nephrectomy and ischemia reperfusion injury and treatment with either saline vehicle or peptide GTI-2 by IV or SC administration is presented in Figure 16. At 3-days post-surgery, the GFR of rats treated with GTI-2 was significantly greater compared with those treated with vehicle. This GTI-2 induced effect on GFR was observed following both IV and SC administration.

[0369] Conclusion

[0370] These data show that in a rat IRI model of AKI, GTI-2 significantly inhibits IRI-mediated kidney injury when dosed by either intravenous or subcutaneous injections over 48 hours as indicated by inhibition of changes in renal function (GFR) and the systemic indicators of renal function, pCreatinine and BUN. Example 7 Intravenous and subcutaneous GTI-2 and GTI-11 pharmacokinetic profile in rats

[0371] Materials and methods

[0372] Male Sprague Dawley rats (250 - 275g) were dosed by either: a single intravenous 2 mL / kg administration of 5 mg / mL GTI-2 or GTI-11 in PBS for a total dose of 10 mg / kg; single subcutaneous 5 mL / kg administration of 5 mg / mL GTI-2 or GTI-11 in PBS for a total dose of 25 mg / kg. Blood was collected via the sub-lingual vein into EDTA tubes at 1 , 5, 10, 15, 30, 60, 120, 240, 360 min post injection. Whole blood (50 pL + 150 pL MQ-water) and plasma (prepared by centrifugation at 2000g for 5 min at 4°C) samples frozen at <-70°C before analysis by LC-MS / MS (Waters Acquity PREMIER UPLC + Waters Xevo TQ-XS triple quadrupole MS with Waters Acquity UPLC Premier Peptide CSH C18 (2.1 x 100 mm, 1.7 pm) column with pre-column filter) using MassLynx 4.2 software. A non-compartmental analysis (NCA) was performed on the data to generate the pharmacokinetic parameters. E.g. Cmax, Tmax, AUC, T1 / 2, etc. using Phoenix WinNonlin from Certara (Canterbury, UK).

[0373] Results

[0374] Single-dose plasma pharmacokinetic parameters of GTI-2 or GTI-11 , administered subcutaneously (SC) or intravenously (IV) are presented in Table 8. IV administration of GTI-2 showed a decreased T1 / 2 and Tmax, and an increased Cmax and AUCinf obs, compared to subcutaneous administration. IV administration of GTI-11 showed a decreased T1 / 2, Cmax and AUCinf obs compared with IV administration of GTI-2.

[0375] Table 8. Single-dose plasma pharmacokinetic parameters of GTI-2 or GTI-11, administered subcutaneously (SC) or intravenously (IV). T1 / 2, half-life; Tmax, time of occurrence of maximum plasma concentration; Cmax, maximum plasma concentration; AUCinf obs, AUC from time of dosing extrapolated to infinity, based on the last observed concentration (_obs).

[0376] Conclusions

[0377] Peptides administered by intravenous route achieve rapid systemic exposure with a terminal half-life that can be extended by subcutaneous administration or adjustment of peptide length and sequence.

[0378] Example 8 Peptide plasma stability

[0379] Materials and methods

[0380] Stability of GTI peptides was determined in human, rat and dog plasma.

[0381] Plasma stability was assessed by incubation for 120 min of each peptide at a final concentration of 1 pM in 400 pL total plasma volume and a final DMSO concentration of 0.5%. Following 0, 20, 40, 60 and 120 min incubation, 40 pL samples were collected a 2-fold volume of acetonitrile with 0.1% trifluoroacetic acid and 6% DMSO was added and samples stored at -20oC until bioanalysis. Peptides were quantified by analysis with LC-MS / MS (Thermo Vanquish Horizon LIHPLC + Thermo Q-Exactive Focus Orbitrap MS with Waters CSH C18 (2.1 x50 mm, 1.7 pm particle size) column with precolumn filter) using Thermo Xcalibur 4.1 .31 .9 software following which stability in terms of extrapolated half-life (t1 / a) and in vitro intrinsic clearance (CLINT) were determined

[0382] Results

[0383] Data demonstrating the stability of GTI-2, GTI- 11 , GTI-65, GTI-79, GTI-83, and GTI-86 in rat, dog and human plasma are presented in Table 9.

[0384] Table 9. Stability of GTI-2, GTI-11, GTI-65, GTI-79, GTI-83, and GTI-86 in rat, dog and human plasma.

[0385] T1 / 2, half-life; CLINT, intrinsic clearance.

[0386] Conclusion

[0387] Substitution of hydrophobic amino acids with hydrophilic amino acids or changing hydrophobic amino acid regions with hydrophilic amino acids to enhance solubility does not negatively impacted the plasma stability of peptides.

[0388] Example 9 Peptide hepatocyte metabolic stability

[0389] Materials and methods

[0390] The metabolic profile and stability of peptides was determined following incubation with either rat, dog or human hepatocytes. Hepatocytes (male SD Rat, viability 66%; male beagle dog, viability 55%; Human mixed gender, viability 79%) at 1 million viable cells / mL were incubated with 1 pM peptide concentration in 300 pL pH7.4 Celsis IVT In Vitro GRO KHB medium with 0.5% DMSO for up to 60 min at 37°C. At either 0, 10, 20, 40 or 60 min incubation 40 pL medium was removed and the incubation terminated with the addition of 2 - fold volume of cold 75% acetonitrile with 0.1% trifluoroacetic acid.

[0391] Samples were analyzed immediately with LC-MS / MS (Waters Aquity LIPLC + Thermo Q-Exactive Orbitrap MS with Waters HSS T3 (2.1 x50 mm, 1 .7 pm particle size) column and using Thermo Xcalibur 4.1 .31 .9 software. Peptide hepatocyte metabolic stability in terms of extrapolated half-life (t1 / a) and in vitro intrinsic clearance (CLINT) were determined, as well as the metabolic profile by assessment of the relative peak profile.

[0392] Results

[0393] Stability parameters of peptides GTI-11 and GTI-2 following incubation with rat, dog or human hepatocytes are presented in Table 10. In rat, dog and human hepatocytes, GTI-2 showed an increased T1 / 2 and decreased CLINT compared with GTI-11 . Relative abundance values of principle metabolites of peptides GTI-11 and GTI-2 following incubation with rat, dog or human hepatocytes are presented in Table 11 . Incubation of hepatocytes with GTI-2 resulted in a greater abundance of native sequence and longer metabolites compared with hepatocytes incubated with GTI-11 . Table 10. Stability parameters of peptides GTI-11 and GTI-2 following incubation with rat, dog or human hepatocytes.

[0394] T1 / 2, half-life; CLINT, intrinsic clearance. Table 11. Relative abundance of principle metabolites of peptides GTI-11 and

[0395] GTI-2 following incubation with rat, dog or human hepatocytes.

[0396] Conclusion

[0397] Hepatocyte metabolism of GTI peptides occurs principally from the C-terminal.

[0398] Amending the C-terminal sequence results in adjusted hepatocyte metabolism with reduced clearance (CLINT), longer t / 2during hepatocyte incubation, and a greater abundance of native sequence and longer metabolites following hepatocyte incubation.

[0399] Example 10 In vivo efficacy of GTI-2 in LPS-induced-acute kidney injury (AKI) Materials & Methods An intraperitoneal (IP) injection of 10 mg / kg lipopolysaccharide (LPS) model of sepsis- induced kidney injury in mice was employed to assess the in vivo activity of peptide GTI-2. Male C57BI 6J mice aged 8-10 weeks (Scanbur, Germany) were injected IP with 10 mg / kg LPS (E. co / / 0111 :B4, L2630, Sigma Aldrich, Soina, Sweden).

[0400] Vehicle (sterile phosphate buffered saline) or GTI-2 formulated in vehicle at 10 mg / mL were administered at 0.5h pre-LPS and 4, and 8 hours post LPS by intravenous IV bolus injection via the tail vein as follows:

[0401] • Peptide GTI-2 stock formulation at 2 mg / mL in PBS, vehicle or peptides dosed at 5 pL / g for a total dose of 10 mg / kg. Following LPS injection, mice were recovered for 24 hours, with blood collected via the tail vein at baseline pre-LPS, 8 and 24 hours post LPS for assessment of kidney injury biomarkers (blood urea nitrogen and pCreatinine), and aspartate amino transferase (AST) as a marker of liver injury. Plasma creatinine (pCreatinine) (Biovision, Milpitas, CA, USA, K625), blood urea nitrogen (BUN) (Thermo Fisher, MA, USA, EIABUN), and AST (MAK055-1 KT, SigmaAldrich, Soina, Sweden) were assessed according to each assay kit manufacturer’s instructions.

[0402] Results

[0403] Mouse pCreatinine, BUN and AST concentrations (mg / dL and nmol / mL / min for AST) and 24-hour LPS kidney and liver injury and treatment with either PBS vehicle or peptide GTI-2 by IV administration is presented in Figure 17. The concentrations of pCreatinine, BUN and AST from mice treated with GTI-2 was significantly lower compared with those treated with vehicle at 24 hours (BUN) and both 8 and 24 hours (pCreatinine & AST) post-LPS treatment.

[0404] Conclusion

[0405] These data show that in a mouse LPS model of kidney and liver injury, GTI-2, significantly inhibits LPS-mediated kidney and liver injury when dosed by IV injections over 24 hours as indicated by inhibition of changes in systemic indicators of renal function (pCreatinine and BUN) and liver injury biomarker, AST. of intravenous or subcutaneous dosed GTI-2 or GTI-86 in

[0406] Materials & Methods

[0407] An intraperitoneal (IP) injection of 20 mg / kg cisplatin model of chemotherapy-induced kidney injury in mice was employed to assess the in vivo activity of peptide GTI-2 and GTI-86.

[0408] Male C57BL / 6 mice (Scanbur, Germany) 8-10 weeks of age were injected with a single dose of 20 mg / kg cisplatin (Sigma, PHR1624), in 10% DMSO in PBS, or vehicle by IP injection of 1 .25 mL / kg body weight per mouse. GTI-2 or IV vehicle control were injected 30 minutes before cisplatin treatment and once daily (every 24h) thereafter by IV bolus injection of 2 mL / kg peptides in PBS, n=6 per group. Both GTI-2 and GTI-86 were dosed IV with 50 pL / 25g body weight (2 mL / kg) of the 5 mg / mL stock via the tail vein to provide 10 mg / kg. A second study tested GTI-86 dosed by subcutaneous (SC) injection of vehicle, or 5, 10 or 20 mg / kg GTI-86 at 30 minutes pre-cisplatin, and daily thereafter. Each SC administration injection split between 2 injection sites was of 8 mL / kg, or 200 pU25g mouse. For 20 mg / kg the stock 2.5 mg / mL formulation was used. For 10 and 5 mg / kg doses, the stock was diluted in PBS to 1 .25 and 0.625 mg / mL respectively. SC injections on the mouse flank used rotating positions to reduce injection site irritancy.

[0409] Following cisplatin injection, mice were recovered for 4 days, with blood collected via the tail vein at baseline pre-cisplatin, and 4 days post cisplatin for assessment of kidney injury biomarkers (blood urea nitrogen and pCreatinine). pCreatinine (pCreatinine) (Biovision, Milpitas, CA, USA, K625), blood urea nitrogen (BUN) (Thermo Fisher, MA, USA, EIABUN) were assessed according to each assay kit manufacturer’s instructions.

[0410] Results

[0411] Mouse pCreatinine and BUN concentrations (mg / dL) and up to 4 day cisplatin-kidney injury and treatment with either PBS vehicle or peptide GTI-2, GTI-86 by IV or SC administration is presented in Figure 18. The concentrations of pCreatinine and BUN from mice treated with GTI-2 and GTI-86 by IV injection were significantly lower compared with those treated with vehicle at 4 days post-cisplatin treatment. GTI-86 when dosed by SC injection produced a dose dependent reduction in both BUN and pCreatinine. Polypeptides GTI-115, GTI-117, GTI-120, GTI-125 & GTI-126 all demonstrated efficacy in the mouse cisplatin-induced kidney injury model.

[0412] Conclusion

[0413] These data show that in a mouse cisplatin model of acute kidney injury, GTI-2 and GTI- 86 dosed by either IV or SC administration routes, significantly inhibit cisplatin- mediated kidney injury as indicated by inhibition of changes in systemic indicators of renal function (pCreatinine and BUN). Polypeptides GTI-115, GTI-117, GTI-120, GTI- 125 & GTI-126 all demonstrated efficacy in the mouse cisplatin-induced kidney injury model. Example 12 In vivo efficacy of subcutaneous dosed A1 M peptide GTI-86 in mouse diabetic kidney disease (DKD) Materials and methods

[0414] A mouse model of streptozotocin (STZ)-induced diabetes and subsequent diabetic kidney disease (DKD) was employed to assess the potential of an A1 M peptide (GTI- 86) to prevent further kidney injury in mice with established diabetes and renal impairment. STZ ablates pancreatic islet cells and induces a model of type 1 diabetes in mice. Diabetes was induced in male 7-8 week old DBA / 2J mice (Janvier, France) by 5 consecutive daily intraperitoneal (IP) injections of STZ (Streptozocin, S0130, Sigma Aldrich, Soina, Sweden) at 40 mg / kg (0.2 mL IP of a 5 mg / mL stock solution in pH4.5 0.1 M citrate acid / sodium citrate buffer), n=8 per group. Baseline blood and urine samples were collected the day before first STZ injection. Non-fasting blood glucose and biomarkers of kidney injury (pCreatinine and BUN) were assessed every 7 days thereafter to determine diabetic phenotype and level of kidney injury. Nineteen days following STZ treatment, when glucose was raised and kidney biomarkers pCreatinine and BUN had shown increases above STZ vehicle levels, treatment with a daily subcutaneous dose of either PBS vehicle or GTI-86 at 0.2, 0.6 or 2 mg / kg (dosed or prepared from an 8 mg / mL stock in PBS at 2 mg / mL) was initiated. Blood and urine were collected at the start of GTI-86 intervention (Day 0), and 14 and 28 days thereafter for urine albumin / creatinine ratio (UACR) and pCreatinine assessment. Albumin and creatinine were assessed using creatinine (K625, Bio Vision, USA) and Albuwell M (Mouse Albumin ELISA) kit (1011 , Ethos Biosciences, USA) as per the manufacturer’s instruction. Blood urea nitrogen (Figure 19D) was assessed using Urea Nitrogen (BUN) Colorimetric Detection Kit (Thermo Fisher, MA, USA, EIABUN).

[0415] Results

[0416] Plasma glucose (non-fasted) was increased in mice treated with STZ from 19 days post STZ treatment (Figure 19A). pCreatinine (Figure 19B) and urine albumin / creatinine ratios (UACR, Figure 19C) increased from baseline to 19 days following STZ treatment (day 0) . Mouse pCreatinine (Figure 19B) and UACR (Figure 19C) were further increased in STZ treated mice from day 0 to 14 and 28 days after initiation of vehicle (PBS) intervention. At both 14- and 28-days post initiation of daily SC GTI-86 treatment, daily GTI-86 peptide administration significantly inhibited STZ- induced increases in pCreatinine, BUN, and UACR in a dose dependent manner. Polypeptides GTI-111 , GTI-115 and GTI-117 also protected mouse kidneys from renal dysfunction in the STZ-diabetic kidney disease model. Polypeptides GTI-111 , GTI-115 and GTI-117 also protected mouse kidneys from renal dysfunction in the STZ-diabetic kidney disease model.

[0417] Conclusion

[0418] These data show that in a mouse STZ model of diabetic kidney disease, A1 M peptide GTI-86, dosed by daily SC administration, significantly inhibited diabetes-mediated kidney injury as indicated by inhibition of changes in systemic indicator of renal function (pCreatinine) and kidney damage (UACR).

[0419] Example 13 In vivo efficacy of subcutaneous dosed A1 M peptide GTI-86 in mouse Adriamvcin-induced model of focal seqmental Glomerulosclerosis (FSGS)

[0420] Materials and methods

[0421] A mouse model of Adriamycin (ADR)-induced focal segmental glomerulosclerosis (FSGS) was employed to assess the potential of A1 M peptide GTI-86 to prevent kidney injury. Structural and functional injury in Adriamycin induced nephropathy mimics chronic kidney disease associated with proteinuria in humans. Male 7-8 week old Balb / c mice (Janvier, France) by a single intravenous (IV) injection or 5 mL / kg of either water for injection vehicle, or Adriamycin (ADR, Sigma Aldrich, St Louis, USA; Doxorubicin hydrochloride D1515) at 10 mg / kg (2.5 mL / kg IV injection of a 4 mg / mL stock solution in water for injection), n=8 per group. Treatment with a daily subcutaneous dose of either PBS vehicle or GTI-86 at 0.2, 0.6, 2 or 6 mg / kg (dosed or prepared from an 8 mg / mL stock in PBS at 2 mg / mL) was initiated 30 minutes prior to Adriamycin challenge (Day 0). Blood and urine were collected at the start of GTI-86 intervention (Day 0), and 9 and 14 days thereafter for urine albumin / creatinine ratio (UACR), pCreatinine and blood urea nitrogen (BUN) assessment. Albumin and creatinine were assessed using creatinine (K625, Bio Vision, USA) and Albuwell M (Mouse Albumin ELISA) kit (1011 , Ethos Biosciences, USA) as per the manufacturer’s instruction. Blood urea nitrogen was assessed using Urea Nitrogen (BUN) Colorimetric Detection Kit (Thermo Fisher, MA, USA, EIABUN).

[0422] Results

[0423] Adriamycin treatment significantly increased pCreatinine, BUN and UACR levels in Adriamycin-treated versus vehicle treated mice at both 9- and 14-days post Adriamycin challenge (Figure 20). The Adriamycin-induced increases in pCreatinine (Figure 20A), BUN (Figure 20B) and UACR (Figure 20C) were all significantly inhibited by daily GTI- 86 treatment in a dose dependent manner. GTI-2, GTI-86, GTI-1 11 , GTI-115 and GTI- 117 all demonstrated inhibition of adriamycin-induced kidney dysfunction.

[0424] Conclusion

[0425] These data indicate that in a mouse Adriamycin model of FSGS, peptide GTI-86, dosed by daily SC administration, significantly inhibited kidney injury as indicated by inhibition of changes in systemic indicators of renal function (pCreatinine) and BUN, as well as kidney damage as indicated by UACR. Polypeptides GTI-1 11 , GTI-1 15 and GTI-1 17 also protected mouse kidneys from renal dysfunction in the STZ-diabetic kidney disease model.

[0426] Example 14 - Peptide design

[0427] The amino acid sequence of human A1 M (Kaumeyer et al., 1986) and three- dimensional crystal structure (Meining and Skerra, 2012) were used to design A1 M- peptides covering the Cys34 position, and various portions of flanking regions. Two peptides were designed and synthesized chemically. The peptides were 36 and 46 amino acids long, respectively and were given names consisting of the three-letter combination of the N-terminal three amino acids followed by the total number of amino acids. The peptides are exact copies of A1 M-sequences. The amino acid sequence and location of the peptides in the A1 M-polypeptide are shown in Fig. 6 and the location of peptide ENF-46 in the three-dimensional A1 M-structure is shown in Fig. 7. As evident from the figures, the peptides are derived from the N-terminal third of the protein and include most of p-strand 1 , loop 1 with the Cys34-position, and p-strand 2. The Cys34 thiol group has been reported to be involved in the reductase, heme-binding and radical-binding activities of A1 M (Allhorn et al., 2005; Rutardottir et al., 2016; Akerstrbm et al., 2007) and was therefore included in both peptides. The sequence motif GKWY (pos. 23-26), which also is included in both peptides for structural reasons, is the first of three conserved lipocalin motifs (MOTIF1 ) which have been suggested to be crucial for the p-barrel construction of the lipocalin fold (Flower, 1996) (however, as shown herein throughout, the motif is not essential for A1 M activity).

[0428] The physico-chemical properties of the peptides are summarized in Fig. 3, and compared to the full-length A1 M-variant, A1 M-035. All parameters were predicted using bioinformatics, except solubility, which was estimated experimentally in PBS pH 7.4, at room-temperature. High solubility >1 mM, medium solubility 0.1-1 mM, low solubility <0.1 mM. As evident from the figure, SRI-36 had limited solubility and may therefore be suboptimal for further studies.

[0429] Example 15 - Determination of size, aooreaation and thiol ciroup activity

[0430] Size and aggregation of the peptide ENF-46 was investigated by SDS-PAGE with or without p-mercaptoethanol (Fig. 8). A strong band was seen at the expected molecular mass, 5 kDa. A weaker band was seen at the double size, approximately 10 kDa, suggesting that the peptide exist in monomeric and dimeric forms, similar to the full- length E.coli A1 M-variants (Fig. 8), and also has been reported for human A1 M isolated from urine (Ekstrom and Berggard, 1977). Less of the dimer and more of the monomer was seen when the thiol-reducing agent p-mercaptoethanol was added to the samples before electrophoresis (Fig. 8, right panel). This shows that the dimer formation partly is mediated by a disulfide bridge between two peptide chains, compatible with redox activity of the cysteine residue.

[0431] Example 16 - Determination of heme bindino

[0432] Heme binding of peptide ENF-46 was investigated by migration shift and tryptophan fluorescence quenching during native PAGE. As seen in Fig. 9 the migration of ENF-46 was considerably faster in the presence of heme, suggesting an increased negative charge. This was also the case for the full-length A1 M variants, although the difference was much smaller than for ENF-46. Moreover, the tryptophan fluorescence of full- length A1 M and the ENF-46 peptide was suppressed in the presence of heme (Fig. 9, right panel). A migration shift towards the anode, and tryptophan fluorescence quenching, were previously shown to be specific traits of recombinant E.coli A1 M (Karnaukhova et al, 2014; Rutardottir et al, 2016). The results thus show that the ENF- 46 peptide is capable of binding heme-groups.

[0433] Heme binding of polypeptides GTI-86, GTI-111 and GTI-115 compared to A1 M-035 was investigated by determination of UV-spectrophotometry absorbance shift (Fig. 21). Absorbance spectra were measured on a NP80 NanoPhotometer (Implen GmbH, Germany) in the UV-Vis region between 240 nm and 700 nm at 22°C. Heme (Applichem, Germany, Hemin porcine A0942) at 40 pM in Tris buffer (10 mM Tris-HCI, pH 8.0) was incubated with A1 M-035 at 45 pM in Tris buffer (10 mM Tris-HCI, pH 8.0) for 120 minutes before scanning UV spectrophotometry in a 96 well plate. Similarly, heme at 40 pM in phosphate buffered saline (pH 7.4) was incubated with peptides at 45 pM in phosphate buffered saline (pH7.4) for 120 minutes before scanning UV spectrophotometry in a 96 well plate. Absorbance spectra were assessed for change in heme absorbance curve, where the heme peak absorbance was 380-390 nm. A1 M- 035 at 45 pM increased the height of the heme absorbance from 1 .78 to 1 .985 indicative of heme binding, as well as shifting the peak from 385 nm to 422 nm, indicating reduction of heme (Fig 21 A). Polypeptide incubation with heme shifted the heme peak absorbance from 1 .42 at 385 nm to 2.04, 1 .87 and 2.09 at 373 nm for (GTI- 86, GTI-11 1 and GTI-115 respectively) indicating polypeptide heme binding (Fig. 21 B- D). The heme binding properties of GTI-86 were maintained with Nle substitution (GTI- 115) and with D-amino acid substitution (GTI-1 11 ).

[0434] When GTI-86 was incubated with 40 pM heme (both in PBS) at increasing concentrations of 45, 90, 180 and 278 pM for 2 hours, there was peptide binding to heme, as evidenced by the increased absorbance peak at 383 nm when heme was incubated with peptide GTI-86 (Fig. 21 E). As GTI-86 concentration increased, there was a increasing absorbance between 400 and 470 nm, corresponding to an increase in reductase activity with higher GTI-86 concentrations (Fig 21 E).

[0435] Example 17 - Determination of cytochrome c reduction

[0436] Reductase activity was investigated by cytochrome c reduction. Enzymatic reduction of cytochrome c in the presence of NADH or NADPH has been reported for human A1 M purified from urine or recombinant Eco / / -produced A1 M (Allhorn et al, 2005). In this report, the reductase activity was completely dependent on the Cys34 thiol group, i.e. no reduction was seen after blocking of the free cysteine with iodoacetamide or by an engineered mutated A1 M-variant, A1 M-C34S, where the Cys34 residue was substituted with a serine residue. As shown in Fig. 10, the appearance of an absorbance peak at 550 nm show that cytochrome c was reduced by both A1 M-wt and the peptide ENF-46 in the presence of NADPH. Similar reduction capacity of a 100 pM cytochrome c / 100 pM NADPH-mixture was displayed by 10 pM full-length A1 M and 20 pM ENF-46 peptide (Fig. 10), as estimated by the height of the 550 nm-peak. Example 18 - Determination of cell protection

[0437] The capacity to protect cells against free heme-induced cell damage was measured using three types of nucleated cell lines and human RBCs (Fig. 11 ). First, K562 cells, a human erythroid cell line grown in suspension was exposed to 100 pM heme in the presence of full-length A1 M, peptide SRI-36 or the control protein ovalbumin for one hour. The cell viability was measured by leakage of LDH into the medium (Fig. 1 1 A). Heme alone induced a massive LDH-leakage, i.e. cell-death. Addition of full-length A1 M (wt and 035 variants) protected against cell damage as expected with significant, but small inhibition at 3 pM and approximately 90% inhibition at 10 pM. This is in accordance with previously published results (Olsson et al., 2008). The peptide SRI-36 showed a small, but significant inhibition of LDH-leakage at 10 and 20 pM, and 80-90% inhibition at 50 pM. The control protein ovalbumin showed no significant inhibition.

[0438] The adherent human kidney (tubular) cell line HK-2 was exposed to different heme concentrations in the presence of 10 pM A1 M-035 or 30 pM ENF-46 peptide for two hours. Cell proliferation, measured by uptake of WST-1 , was suppressed in a dosedependent manner to increasing concentrations of heme. At all concentrations, AI M- 035 and ENF-46 reversed the heme-induced suppression of cell proliferation (Fig. 1 1 B). Using 30 pM heme, the effects of dilution series of A1 M-035, ENF-46 and the non-A1 M reference peptide SWT-21 were measured after two hours incubation. A dose-dependent significant inhibition was obtained with A1 M-035 and ENF-46 but not SWT-21 (Fig. 11 C).

[0439] Protective effects of ENF-46 were also seen on the adherent human hepatocyte cell line HepG2, measured by heme-induced LDH-leakage after one hour incubation. As shown in Fig. 11 D, 30 and 60 pM ENF-46 yielded significant, almost complete inhibition of the heme-induced LDH-leakage, similar to 10 pM full-length A1 M-035. Small, but significant inhibition was also seen by 7.5 and 15 pM ENF-46.

[0440] A1 M was recently shown to protect RBC from heme-induced cell damage, i.e. hemolysis (Kristiansson et al., 2020). Here, we could show that this was also achieved by the peptide ENF-46. Fig. 11 E shows the effects of a dilution series of ENF-46 (1 .25- 50 pM) on LDH-leakage from RBC incubated with 30 pM heme for three hours.

[0441] Significant inhibition of heme-induced hemolysis is seen above 25 pM ENF-46. Sequence overview

[0442] GTI-1 (A1 M fragment 1 ): C34 (SEQ ID NO: 1)

[0443] DDDDKQVQENFDISRIYGKWYNLAIGSTCPWLKKIMDRMTVSTLVLGEGAT

[0444] GTI-2 (A1 M fragment 2): C34 (SEQ ID NO: 2)

[0445] DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGAT

[0446] GTI-3 (A1 M fragment 3): C34 (SEQ ID NO: 3)

[0447] DDKGPVPTPPDNIQVQENFDISRIYGKWYNLAIGSTCPWLKKIM

[0448] GTI-4 (A1 M fragment 4): C34 (SEQ ID NO: 4) KGSTCPWLKKIMDRMTVSTLVLGEGAT

[0449] GTI-5 (A1 M scrambled sequence) (SEQ ID NO: 5)

[0450] MGSEDLMETVWYKIAEGITGPRKELNKLLETSATV

[0451] GTI-6 (A1 M fragment 6): C34 and C72 (SEQ ID NO: 6)

[0452] GSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTHWRKGVCEETSGAYEK

[0453] GTI-7 (A1 M fragment 7): C34 (SEQ ID NO: 7)

[0454] DDDDKGPVPTPPDNIQVQENFDISRIYGKWYNLAIGSTCPWLKKIMDRM

[0455] GTI-8 (A1 M fragment 8): C34 and C72 (SEQ ID NO: 8)

[0456] GSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTHWRKGVCEET

[0457] GTI-9 (A1 M fragment 9): C34 (SEQ ID NO: 9)

[0458] GSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTHWRK

[0459] GTI-10 (A1 M fragment 10): C34 (SEQ ID NO: 10)

[0460] GSTCPWLKKIMDRMTVSTLVLGEGATEAEISMT

[0461] GTI-11 (A1 M fragment 11): C34 (SEQ ID NO: 11 )

[0462] GSTCPWLKKIMDRMTVSTLVLGEG

[0463] GTI-12 (A1 M fragment 12): C34 (SEQ ID NO: 12) NLAIGSTCPWLKKIMDR

[0464] GTI-13 (A1 M fragment 13): C72 (SEQ ID NO: 13)

[0465] GEGATEAEISMTSTHWRKGVCEETSGAYEKTDTDG

[0466] GTI-14 (A1 M fragment 14): C72 (SEQ ID NO: 14)

[0467] GATEAEISMTSTHWRKGVCEETSGAYEKTD

[0468] GTI-15 (A1 M fragment 15): no cysteine (SEQ ID NO: 15) EKTDTDGKFLYHKSKWDITMESYVV

[0469] M8H4DK-tagged A1 M mutant (SEQ ID NO: 16)

[0470] MHHHHHHHHDDDDKGPVPTPPDNIQVQENFDISRIYGKWYNLAIGSTCPWLKKIMDR

[0471] MTVSTLVLGEGATEAEISMTSTHWRKGVCEETSGAYEKTDTDGKFLYHKSKWDITME

[0472] SYVVHTNYDEYAIFLTKKFSRHHGPTITAKLYGRAPQLRETLLQDFRVVAQGVGIPEDS

[0473] IFTMADRGECVPGEQEPEPILIPR

[0474] M8H4DK-tagged A1 M (SEQ ID NO: 17)

[0475] MHHHHHHHHDDDDKGPVPTPPDNIQVQENFNISRIYGKWYNLAIGSTCPWLKKIMDR

[0476] MTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDGKFLYHKSKWNITME

[0477] SYVVHTNYDEYAIFLTKKFSRHHGPTITAKLYGRAPQLRETLLQDFRVVAQGVGIPEDS

[0478] IFTMADRGECVPGEQEPEPILIPR

[0479] Truncated, M8H4DK-tagged A1 M mutant (SEQ ID NO: 18)

[0480] MHHHHHHHHDDDDKGPVPTPPDNIQVQENFDISRIYGKWYNLAIGSTCPWLKKIMDR

[0481] MTVSTLVLGEGATEAEISMTSTHWRKGVCEETSGAYEKTDTDGKFLYHKSKWDITME

[0482] SYVVHTNYDEYAIFLTKKFSRHHGPTITAKLYGRAPQLRETL

[0483] Truncated, M8H4DK-tagged A1 M (SEQ ID NO: 19)

[0484] MHHHHHHHHDDDDKGPVPTPPDNIQVQENFNISRIYGKWYNLAIGSTCPWLKKIMDR

[0485] MTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDGKFLYHKSKWNITME

[0486] SYVVHTNYDEYAIFLTKKFSRHHGPTITAKLYGRAPQLRETL

[0487] A1 M fragment - C34 (SEQ ID NO: 20)

[0488] GSTCPWLKKIX1 A1 M fragment - C72 (SEQ ID NO: 21 )

[0489] GATEAEISMTSTX2WRKGVCEETSGAYEKTD

[0490] A1 M fragment - C34 + X1is M (SEQ ID NO: 22)

[0491] GSTCPWLKKIM

[0492] A1 M fragment - C34 + X1is K (SEQ ID NO: 23) GSTCPWLKKIK

[0493] A1 M fragment - C34 + X1is R (SEQ ID NO: 24)

[0494] GSTCPWLKKIR

[0495] A1 M fragment - C72 + X2is H (SEQ ID NO: 25)

[0496] GATEAEISMTSTHWRKGVCEETSGAYEKTD

[0497] A1 M fragment - C72 + X2is R (SEQ ID NO: 26) GATEAEISMTSTRWRKGVCEETSGAYEKTD

[0498] A1 M fragment - C72 + X2is K (SEQ ID NO: 27) GATEAEISMTSTKWRKGVCEETSGAYEKTD

[0499] M8H4DK-tagged A1 M variant sequence - X3is D, N, or G; X4is M, K, R, Nle; X5is H, R, or K, X6is D, N, or E, X7is M or Nle (SEQ ID NO: 28)

[0500] MHHHHHHHHDDDDKGPVPTPPDNIQVQENFX3ISRIYGKWYNLAIGSTCPWLKKIX4D

[0501] RX7TVSTLVLGEGATEAEISMTSTX5WRKGVCEETSGAYEKTDTDGKFLYHKSKWX6IT

[0502] MESYVVHTNYDEYAIFLTKKFSRHHGPTITAKLYGRAPQLRETLLQDFRVVAQGVGIP EDSIFTMADRGECVPGEQEPEPILIPR

[0503] Truncated M8H4DK-tagged A1 M variant sequence - X3is D, N, or G; X4is M, K, R, or Nle; X5is H, R, or K, X6is D, N, or E, X7is M or Nle (SEQ ID NO: 29)

[0504] MHHHHHHHHDDDDKGPVPTPPDNIQVQENFX3ISRIYGKWYNLAIGSTCPWLKKIX4D

[0505] RX7TVSTLVLGEGATEAEISMTSTX5WRKGVCEETSGAYEKTDTDGKFLYHKSKWX6IT

[0506] MESYVVHTNYDEYAIFLTKKFSRHHGPTITAKLYGRAPQLRETL Human A1 M (SEQ ID NO: 30)

[0507] GPVPTPPDNIQVQENFNISRIYGKWYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATEA

[0508] EISMTSTRWRKGVCEETSGAYEKTDTDGKFLYHKSKWNITMESYVVHTNYDEYAIFLT

[0509] KKFSRHHGPTITAKLYGRAPQLRETLLQDFRVVAQGVGIPEDSIFTMADRGECVPGEQ EPEPILIPR

[0510] GTI-16 (SEQ ID NO: 31)

[0511] GSTCPWLKKIMDRMTVSTLDLGEG

[0512] GTI-17 (SEQ ID NO: 32)

[0513] GSTCPWLKKIMDRMTVSTLSLGEG

[0514] GTI-18 (SEQ ID NO: 33)

[0515] GSTCPWLKKIMDRMTVSTLVLGEGATE

[0516] GTI-24 - PEGylation GTI-11 (SEQ ID NO: 34)

[0517] GSTCPWLKKIMDRMTVSTLVLGEG -PEG2-CONH2

[0518] GTI-25 - PEGylation GTI-16 (SEQ ID NO: 35)

[0519] GSTCPWLKKIMDRMTVSTLDLGEG -PEG2-CONH2

[0520] GTI-26 - Albumin binding domain (SEQ ID NO: 36)

[0521] GSTCPWLKKI M DRMTVSTLVLG EGSDFYKRLINKAKTVEGVEALKLHILAALP

[0522] GTI-27 - Albumin binding domain (SEQ ID NO: 37)

[0523] GSTCPWLKKIMDRMTVSTLVLGEGGGGGSASSDFYKRLINKAKTVEGVEALKLHILAA LP

[0524] GTI-28 - Albumin binding domain (SEQ ID NO: 38)

[0525] GSTCPWLKKIMDRMTVSTLVLGEGGGGGSGGGGSGGGGSASSDFYKRLINKAKTVE

[0526] GVEALKLHILAALP

[0527] GTI-29 - PEGylation GTI-12 (SEQ ID NO: 39)

[0528] AC-NLAIGSTCPWLKKIMDR-CONH2-PEG2 GTI-30 - PEGylation GTI-2 (SEQ ID NO: 40)

[0529] AC-DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGAT-PEG2

[0530] GTI-31 - Albumin Binding Sequence GTI-12 (SEQ ID NO: 41 )

[0531] N LAIGSTCPWLKKIMDRGGGGSASSDFYKRLINKAKTVEGVEALKLHILAALP

[0532] GTI-32 - Albumin Binding Sequence GTI-12 (SEQ ID NO: 42)

[0533] NLAIGSTCPWLKKIMDRGGGGSGGGGSGGGGSASSDFYKRLINKAKTVEGVEALKLH ILAALP

[0534] GTI-33 - Albumin Binding Sequence GTI-12 (SEQ ID NO: 43)

[0535] DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATGGGGSASSDFYKRLINKAKTV EGVEALKLHILAALP

[0536] GTI-34 - Albumin Binding Sequence GTI-12 (SEQ ID NO: 44)

[0537] DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATGGGGSGGGGSGGGGSASSD

[0538] FYKRLINKAKTVEGVEALKLHILAALP

[0539] GTI-35 - GTI-11 TFA salt (SEQ ID NO: 45)

[0540] GSTCPWLKKIMDRMTVSTLVLGEG

[0541] GTI-36 - GTI-11 chloride salt (SEQ ID NO: 46)

[0542] GSTCPWLKKIMDRMTVSTLVLGEG

[0543] GTI-37 - GTI-11 (SS-Dimer) (SEQ ID NO: 47)

[0544] GSTCPWLKKIMDRMTVSTLVLGEG

[0545] GTI-38 - GTI-11 cysteine modification Cam (SEQ ID NO: 48)

[0546] GSTC(Cam)PWLKKIMDRMTVSTLVLGEG

[0547] GTI-39 - GTI-11 cysteine modification homocysteine (SEQ ID NO: 49) GST{hC}PWLKKIMDRMTVSTLVLGEG

[0548] GTI-40 - GTI-11 cysteine modification penicillamine (SEQ ID NO: 50) GST{Penc}PWLKKIMDRMTVSTLVLGEG GTI-41 - GTI-11 methionine sulfoxide x2 (SEQ ID NO: 51 )

[0549] GSTCPWLKKIM(ox)DRM(ox)TVSTLVLGEG

[0550] GTI-42 - GTI-11 truncated (SEQ ID NO: 52) GSTC

[0551] GTI-43 - GTI-11 truncated (SEQ ID NO: 53) CPWLKKIMDR

[0552] GTI-44 - GTI-11 backbone CLIPs (SEQ ID NO: 54) C1 -GSTCPWLKKIMDR-C1 -TVSTLVLGEG

[0553] GTI-45 - GTI-11 backbone CLIPs (SEQ ID NO: 55) C1 -NLAIGSTCPWLKKIMDRMTVS-C1

[0554] GTI-46 - GTI-11 backbone CLIPs (SEQ ID NO: 56) C1 -AIGSTCPWLKKIMDRMT-C1

[0555] GTI-48 - scramble with cysteine (SEQ ID NO: 57)

[0556] H-LRGCGMSLTVPSWTLKDKVEITGM-OH

[0557] GTI-49 - scramble with cysteine (SEQ ID NO: 58)

[0558] H-PWSCLTKLVKLIMGEDGRGMTSVT-OH

[0559] GTI-50 - GTI-12 backbone CLIPs (SEQ ID NO: 59) C1 -NLAIGSTCPWLKKIMDR-C1

[0560] GTI-52 - GTI-12 PEG5 (SEQ ID NO: 60)

[0561] NLAIGSTCPWLKKIMDR-PEG5

[0562] GTI-53 - GTI-12 PEG10 (SEQ ID NO: 61 )

[0563] NLAIGSTCPWLKKIMDR-PEG10

[0564] GTI-54 - GTI-11 PEG5 (SEQ ID NO: 62) GSTCPWLKKIMDRMTVSTLVLGEG-PEG5

[0565] GTI-55 - GTI-11 PEG10 (SEQ ID NO: 63)

[0566] GSTCPWLKKIMDRMTVSTLVLGEG-PEG1 0

[0567] GTI-56 - GTI-12 PEG20 (SEQ ID NO: 64)

[0568] NLAIGSTCPWLKKIMDR-PEG20

[0569] GTI-57 - GTI-11 PEG20 (SEQ ID NO: 65)

[0570] GSTCPWLKKIMDRMTVSTLVLGEG-PEG20

[0571] GTI-58 - GTI-12 D-Amino acid substitution (SEQ ID NO: 66) NLAIGSTCP[D-Trp]LKKIMDR

[0572] GTI-59 - GTI-12 D-Amino acid substitution (SEQ ID NO: 67) NLAIGSTCPW[D-Leu]KKIMDR

[0573] GTI-60 - GTI-12 D-Amino acid substitution (SEQ ID NO: 68) NLAIGSTCPWLK[D-Lys]IMDR

[0574] GTI-61 - GTI-11 D-Amino acid substitution (SEQ ID NO: 69)

[0575] GSTCPWLK[D-Lys]IMDRMTVSTLVLGEG

[0576] GTI-62 - GTI-11 D-Amino acid substitution (SEQ ID NO: 70)

[0577] GSTCPWLKKIMD[D-Arg]MTVSTLVLGEG

[0578] GTI-63 - GTI-11 D-Amino acid substitution (SEQ ID NO: 71)

[0579] GSTCPWLKKIMDRMTVSTLV[D-Leu]GEG

[0580] GTI-64 - GTI-11 D-Amino acid substitution (SEQ ID NO: 72)

[0581] GSTCPWLKKIMDRMTVSTL[D-Asp]LGEG

[0582] GTI-65 - GTI-11 Arginine side chain methylation (SEQ ID NO: 73) GSTCPWLKKIMD[Arg methylated side chain]MTVSTLVLGEG GTI-66 - GTI-11 Lysine dimethylation (SEQ ID NO: 74) GSTCPWLK[Lys di-methylated]IMDRMTVSTLVLGEG

[0583] GTI-67 - GTI-11 Threonine N-methylation (SEQ ID NO: 75)

[0584] GSTCPWLKKIMDRM[Thr N-Methylated]VSTLVLGEG

[0585] GTI-68 - GTI-11 Isoleucine N-methylation (SEQ ID NO: 76) GSTCPWLKK[lle N-methylated]MDRMTVSTLVLGEG

[0586] GTI-69 - GTI-12 side chain methylation (SEQ ID NO: 77)

[0587] NLAIGSTCPWLK[Lys di-methylated]IMDR

[0588] GTI-70 - GTI-12 Threonine N-methylation (SEQ ID NO: 78)

[0589] NLAIGS[Thr N-Methylated]CPWLKKIMDR

[0590] GTI-71 - GTI-12 Isoleucine N-methylation (SEQ ID NO: 79)

[0591] NLA[lle N-methylated]GSTCPWLKKIMDR

[0592] GTI-72 - GTI-12 Arginine side chain methylation (SEQ ID NO: 80)

[0593] NLAIGSTCPWLKKIMD[Arg methylated side chain]

[0594] GTI-73 - GTI-11 D-amino acid (SEQ ID NO: 81 )

[0595] G[D-Ser]TCPWLKKIMDRMTVSTLVLGEG

[0596] GTI-74 - GTI-11 D-amino acid (SEQ ID NO: 82) [D-Asn]LAIGSTCPWLKKIMDR

[0597] A1 M fragment pos 1 -92 of human wtA1 M (SEQ ID NO: 83)

[0598] GPVPTPPDNIQVQENFNISRIYGKWYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATEA

[0599] El SMTSTRWRKGVCEETSGAYE KTDTDGKFLYHK

[0600] A1 M fragment pos 22-92 of human wtA1 M (SEQ ID NO: 84)

[0601] YGKWYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGA

[0602] YEKTDTDGKFLYHK A1 M fragment: pos 22-62 of human wtA1 M (SEQ ID NO: 85)

[0603] YGKWYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATEAEISM

[0604] A1 M fragment: pos 26-62 of human wtA1 M (SEQ ID NO: 86)

[0605] YNLAIGSTCPWLKKIMDRMTVSTLVLGEGATEAEISM

[0606] A1 M fragment: pos 30-62 of human wtA1 M (SEQ ID NO:87) IGSTCPWLKKIMDRMTVSTLVLGEGATEAEISM

[0607] A1 M fragment: pos 31-56 of human wtA1 M (SEQ ID NO: 88) GSTCPWLKKIMDRMTVSTLVLGEGAT

[0608] A1 M fragment: pos 26-56 of human wtA1 M (SEQ ID NO: 89) YNLAIGSTCPWLKKIMDRMTVSTLVLGEGAT

[0609] A1 M fragment: pos 26-92 of human wtA1 M (SEQ ID NO: 90)

[0610] YNLAIGSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKT DTDGKFLYHK

[0611] A1 M fragment: pos 30-92 of human wtA1 M (SEQ ID NO: 91 )

[0612] IGSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDG KFLYHK

[0613] A1 M fragment: pos 31-92 of human wtA1 M (SEQ ID NO: 92)

[0614] GSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDGK FLYHK

[0615] N-terminal tag 8H5GIEGR to SEQ ID NOs: 30, 83-92, 100-116 (SEQ ID NO: 93) HHHHHHHHGGGGGIEGR

[0616] N-terminal tag 8H4DK to SEQ ID NOs: 30, 83-92, 100-116 (SEQ ID NO: 94) HHHHHHHHDDDDK

[0617] N-terminal tag 6H4DK to SEQ ID NOs: 30, 83-92, 100-116 (SEQ ID NO: 95)

[0618] HHHHHHDDDDK N / C-terminal tag 8H to SEQ ID NOs: 30, 83-92, 100-116 (SEQ ID NO: 96)

[0619] HHHHHHHH

[0620] C-terminal tag RGIE5G8H to SEQ ID NOs: 30, 83-92, 100-116 (SEQ ID NO: 97) RGIEGGGGGHHHHHHHH

[0621] C-terminal tag K4D8H to SEQ ID NOs: 30, 83-92, 100-116 (SEQ ID NO: 98) KDDDDHHHHHHHH

[0622] C-terminal tag K4D6H to SEQ ID NOs: 30, 83-92, 100-116 (SEQ ID NO: 99) KDDDDHHHHHH

[0623] A1 M fragment: pos 23-91 of human wtA1 M (SEQ ID NO: 100)

[0624] GKWYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAY EKTDTDGKFLYH

[0625] A1 M fragment: pos 23-33 of human wtA1 M (SEQ ID NO: 101)

[0626] GKWYNLAIGST

[0627] A1 M fragment: pos 35-91 of human wtA1 M (SEQ ID NO: 102)

[0628] PWLKKIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDGKFLYH

[0629] SRI-36: pos 19-54 of human wtA1 M (SEQ ID NO: 103)

[0630] SRIYGKWYNLAIGSTCPWLKKIMDRMTVSTLVLGEG

[0631] ENF-46: pos 14-59 of human wtA1 M (SEQ ID NO: 104)

[0632] ENFNISRIYGKWYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATEAE

[0633] A1 M fragment: pos 22-183 of human wtA1 M (SEQ ID NO: 105)

[0634] YGKWYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGA

[0635] YEKTDTDGKFLYHKSKWNITMESYVVHTNYDEYAIFLTKKFSRHHGPTITAKLYGRAP

[0636] QLRETLLQDFRVVAQGVGIPEDSIFTMADRGECVPGEQEPEPILIPR

[0637] A1 M fragment: pos 22-160 of human wtA1 M (SEQ ID NO: 106) YGKWYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGA

[0638] YEKTDTDGKFLYHKSKWNITMESYVVHTNYDEYAIFLTKKFSRHHGPTITAKLYGRAP

[0639] QLRETLLQDFRVVAQGVGIPEDSI

[0640] A1 M fragment: pos 51-155 of human wtA1 M (SEQ ID NO: 107)

[0641] LGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDGKFLYHKSKWNITMESYVVHTN

[0642] YDEYAIFLTKKFSRHHGPTITAKLYGRAPQLRETLLQDFRVVAQGVGI

[0643] A1 M fragment: pos 59-135 of human wtA1 M (SEQ ID NO: 108)

[0644] EISMTSTRWRKGVCEETSGAYEKTDTDGKFLYHKSKWNITMESYVVHTNYDEYAIFLT KKFSRHHGPTITAKLYGRA

[0645] A1 M fragment: pos 92-183 of human wtA1 M (SEQ ID NO: 109)

[0646] KSKWNITMESYVVHTNYDEYAIFLTKKFSRHHGPTITAKLYGRAPQLRETLLQDFRVVA

[0647] QGVGIPEDSIFTMADRGECVPGEQEPEPILIPR

[0648] A1 M fragment: pos 22-135 of human wtA1 M (SEQ ID NO: 110)

[0649] YGKWYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGA

[0650] YEKTDTDGKFLYHKSKWNITMESYVVHTNYDEYAIFLTKKFSRHHGPTITAKLYGRA

[0651] A1 M fragment: pos 26-135 of human wtA1 M (SEQ ID NO: 111 )

[0652] YNLAIGSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKT

[0653] DTDGKFLYHKSKWNITMESYVVHTNYDEYAIFLTKKFSRHHGPTITAKLYGRA

[0654] A1 M fragment: pos 30-135 of human wtA1 M (SEQ ID NO: 112)

[0655] IGSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDG

[0656] KFLYHKSKWNITMESYVVHTNYDEYAIFLTKKFSRHHGPTITAKLYGRA

[0657] A1 M fragment: pos 31-135 of human wtA1 M (SEQ ID NO: 113)

[0658] GSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDGK

[0659] FLYHKSKWNITMESYVVHTNYDEYAIFLTKKFSRHHGPTITAKLYGRA

[0660] A1 M fragment: pos 61-135 of human wtA1 M (SEQ ID NO: 114)

[0661] SMTSTRWRKGVCEETSGAYEKTDTDGKFLYHKSKWNITMESYVVHTNYDEYAIFLTK

[0662] KFSRHHGPTITAKLYGRA A1 M fragment: pos 66-135 of human wtA1 M (SEQ ID NO: 115) RWRKGVCEETSGAYEKTDTDGKFLYHKSKWNITMESYVVHTNYDEYAIFLTKKFSRH HGPTITAKLYGRA

[0663] A1 M fragment: pos 87-135 of human wtA1 M (SEQ ID NO: 116) KFLYHKSKWNITMESYVVHTNYDEYAIFLTKKFSRHHGPTITAKLYGRA

[0664] A1 M fragment: pos 92-135 of human wtA1 M (SEQ ID NO: 117) KSKWNITMESYVVHTNYDEYAIFLTKKFSRHHGPTITAKLYGRA

[0665] Albumin binding domain (ABD1) (SEQ ID NO: 118)

[0666] LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP

[0667] Albumin binding domain fragment (SEQ ID NO: 119) SDFYKRLINKAKTVEGVEALKLHILAALP wt A1 M (SEQ ID NO: 120)

[0668] GPVPTPPDNIQVQENFNISRIYGKWYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATEA EISMTSTRWRKGVCEETSGAYEKTDTDGKFLYHKSKWNITMESYVVHTNYDEYAIFLT KKFSRHHGPTITAKLYGRAPQLRETLLQDFRVVAQGVGIPEDSIFTMADRGECVPGEQ EPEPILIPR

[0669] Linker (SEQ ID NO: 121)

[0670] GGGGGSAS

[0671] Linker (SEQ ID NO: 122)

[0672] GGGGSGGGGSGGGGSAS

[0673] GTI-77 (SEQ ID NO: 123)

[0674] GSTCPWLKKIMDRM[D-Thr]VSTLVLGEG

[0675] GTI-78 (SEQ ID NO: 124)

[0676] GSTCPWLKKIMDRMT[D-Val]STLVLGEG GTI-79 (SEQ ID NO: 125)

[0677] GSTCPWLKKIMDRM[D-Thr][D-Val]STLVLGEG

[0678] GTI-82 (SEQ ID NO: 126)

[0679] GSTCPWLKKIMDRMTESTLVLGEG

[0680] GTI-83 (SEQ ID NO: 127)

[0681] GSTCPWLKKIMDRMTKSTLVLGEG

[0682] GTI-84 (SEQ ID NO: 128)

[0683] EEEEKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGAT

[0684] GTI-85 (SEQ ID NO: 129)

[0685] EEKYNLAIGSTCPWLKKIMDRMTVSTLVLGE

[0686] GTI-86 (SEQ ID NO: 130)

[0687] EEKYNLAIGSTCPWLKKIMDRMTESTLVLGE

[0688] Peptides 75, 76, 87, 88, 89, 90 have a full-length albumin binding domain. 75 & 76 are GTI-11 with an ABD1 (SEQ ID NO: 118) on the N- and C- terminals respectively. They have a GGGGGSAS linking motif

[0689] GTI-75 (SEQ ID NO: 131 )

[0690] LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALPGGGGGSAS

[0691] GSTCPWLKKIMDRMTVSTLVLGEG

[0692] GTI-76 (SEQ ID NO: 132)

[0693] GSTCPWLKKIMDRMTVSTLVLGEGGGGGGSASLAEAKVLANRELDKYGVSDFYKRLI NKAKTVEGVEALKLHILAALP

[0694] Peptides 87 and 88 also have N-terminal full-length ABD1 with linker, conjugated to peptide GTI-82, GTI-87 has a single linker and GTI-82, GTI-88 has the linker and GTI- 82 repeated 3 times.

[0695] GTI-87 (SEQ ID NO: 133) LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALPGGGGGSAS GSTCPWLKKIMDRMTESTLVLGEG

[0696] GTI-88 (SEQ ID NO: 134) LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALPGGGGGSASGSTC PWLKKIMDRMTESTLVLGEGGGGGGSASGSTCPWLKKIMDRMTESTLVLGEGGGG

[0697] GGSASGSTCPWLKKIMDRMTESTLVLGEG

[0698] Peptides 89 and 90 also have N-terminal full-length ABD1 with linker, conjugated to peptide GTI-86, GTI-89 has a single linker and GTI-86, GTI-90 has the linker and GTI- 86 repeated 3 times.

[0699] GTI-89 (SEQ ID NO: 135)

[0700] LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALPGGGGGSAS

[0701] EEKYNLAIGSTCPWLKKIMDRMTESTLVLGE

[0702] GTI-90 (SEQ ID NO: 136)

[0703] LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALPGGGGGSASEEKY NLAIGSTCPWLKKIMDRMTESTLVLGEGGGGGSASEEKYNLAIGSTCPWLKKIMDRM TESTLVLGEGGGGGSASEEKYNLAIGSTCPWLKKIMDRMTESTLVLGE

[0704] SEQ ID NO: 137

[0705] GKWYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTRWR

[0706] Hu lgG1 Fc fragment (SEQ ID NO: 138)

[0707] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0708] GTI-97 (SEQ ID NO:139)

[0709] GSTCPWLKKI[Nle]DR[Nle]TVSTLVLGEG GTI-98 (SEQ ID NQ:140)

[0710] GSTCPWLKKI[Nle]DR[Nle]TVSTL[D-Asp]LGEG

[0711] GTI-99 (SEQ ID NO:141)

[0712] GSTCPWLKKI[Nle]DR[Nle]TVSTLVLGEG[D-Asp][D-Thr][D-Glu]

[0713] GTI-100 (SEQ ID NO:142)

[0714] GSTCPWLKKI[Nle]DR[Nle]TKSTLVLGEG

[0715] GTI-101 (SEQ ID NO:143)

[0716] EEKYNLAIGSTCPWLKKI[Nle]DR[Nle]TVSTLVLGE

[0717] GTI-102 (SEQ ID NO:144)

[0718] GSTCPWLKKI[Nle]DR[Nle]TVSTL[D-Asp]LGEG

[0719] A1 M fragment - C34 + X1is Nle (SEQ ID NO:145) GSTCPWLKKINIe

[0720] GTI-111 (SEQ ID NO:146)

[0721] GSTCPWLKKIMDRMTKSTL[d-D]LGEG

[0722] GTI-112 (SEQ ID NO:147)

[0723] EEKYNLAIGSTCPWLKKIMDRMTESTLDLGE

[0724] GTI-113 (SEQ ID NO:148)

[0725] EEKYNLAKGSTCPWLKKIMDRMTESTLDLGE

[0726] GTI-114 (SEQ ID NO:149)

[0727] GSTCPWLKKI[NLe]DR[NLe]TKSTL[d-D]LGEG

[0728] GTI-115 (SEQ ID NO:150)

[0729] EEKYNLAIGSTCPWLKKI[NLe]DR[NLe]TESTLDLGE

[0730] GTI-116 (SEQ ID NO:151)

[0731] EEKYNLAKGSTCPWLKKI[NLe]DR[NLe]TESTLDLGE GTI-117 (SEQ ID NO:152)

[0732] GSTCPWLKKI[NLe]DR[NLe]TK[N-Meth S]TL[D]LGEG

[0733] GTI-118 (SEQ ID NO:153)

[0734] EEKYNLAIGSTCPWLKKI[NLe]DR[NLe]TE[N-Meth SJTLDLGE

[0735] GTI-119 (SEQ ID NO:154)

[0736] EEKYNLAKGSTCPWLKKI[NLe]DR[NLe]TE[N-Meth SJTLDLGE

[0737] GTI-120 (SEQ ID NO:155)

[0738] Ac-EEKYNLAIGSTCPWLKKI[Nle]DR[Nle]TKSTLVLGE-NH2

[0739] GTI-121 (SEQ ID NO:156 - ([C20DA]-[yGlu]-[OEG]-[OEG]) - SEQ ID NO:164)

[0740] Ac-EEKYNLAIGSTCPWLKKI[Nle]DR[Nle]T[K([C20DA]-[yGlu]-[OEG]-[QEG]- )]STLVLGE-NH2

[0741] GTI-122 (SEQ ID NO:157) [C20DA]-[yGlu]-[OEG]-[QEG]-EEKYNLAIGSTCPWLKKI[Nle]DR[Nle]TKSTLVLGE-NH2

[0742] GTI-123 (SEQ ID NO:158 - ([C20DA]-[yGlu]-[OEG]-[OEG]) - SEQ ID NO:165)

[0743] Ac-GSTCPWLK[K([C20DA]-[yGlu]-[OEG]-[QEG]-

[0744] )]l[NLe]DR[NLe]TK[NMeSer]TL[D]LGEG-NH2

[0745] GTI-124 (SEQ ID NO:159) [C20DA]-[yGlu]-[OEG]-[QEG]-GSTCPWLKKI[NLe]DR[NLe]TK[NMeSer]TL[D]LGEG- NH2

[0746] GTI-125 (SEQ ID NQ:160) (Head to tail cyclization of GTI-120. C-terminal Cysteine added to facilitate cyclization, cyclization achieved by reaction of C-terminal cysteine thiol group with N-terminal chloroacetate)

[0747] Cyclo(CIAc-EEKYNLAIGSTCPWLKKI[Nle]DR[Nle]TKSTLVLGE-Cys)-NH2

[0748] GTI-126 (SEQ ID NO:161)

[0749] Ac-EEKYNLAIGSTCPWLKKI[Nle]DR[Nle]TESTLVLGE-NH2 GTI-127 (SEQ ID NO:162) (Head to tail cyclization of GTI-86. C-terminal Cysteine added to facilitate cyclization) Cyclo(CIAc-EEKYNLAIGSTCPWLKKIMDRMTESTLVLGE-Cys)-NH2

[0750] GTI-128 (SEQ ID NO:163) GTI-86 with C-terminal albumin binding domain sequence Ac- EEKYNLAIGSTCPWLKKIMDRMTESTLVLGEGGGGGSASLAEAKVLANRELDKYGVS DFYKRLINKAKTVEGVEALKLHILAALP-NH2

[0751] References

[0752] Allhorn M, Klapyta A, Akerstrbm B. Redox properties of the lipocalin alphal - microglobulin: reduction of cytochrome c, hemoglobin, and free iron. Free Radio. Biol. Med. 2005 Mar 1 ;38(5):557-67.

[0753] Ekstrom B, Berggard I. Human alphal -microglobulin. Purification procedure, chemical and physiochemical properties. J Biol. Chem. 1977 Nov 25;252(22):8048-57.

[0754] Flower, Biochem J. 1996 Aug 15;318 ( Pt 1 )(Pt 1 ):1 -14.

[0755] Karnaukhova, E. etal., , Front Physiol, 2014, 5, p.465.

[0756] Kaumeyer JF, Polazzi JO, Kotick MP. The mRNA for a proteinase inhibitor related to the HI-30 domain of inter-alpha-trypsin inhibitor also encodes alpha-1 -microglobulin (protein HC). Nucleic Acids Res. 1986 Oct 24;14(20):7839-50.

[0757] Kristiansson A, Bergwik J, Alattar AG, Flygare J, Gram M, Hansson SR, Olsson ML, Storry JR, Allhorn M, Akerstrbm B. Human radical scavenger a1 -microglobulin protects against hemolysis in vitro and a1 -microglobulin knockout mice exhibit a macrocytic anemia phenotype. Free Radic Biol. Med. 2020 Feb 21 :S0891 -5849(19)32350-0.

[0758] Meining W, Skerra A. The crystal structure of human a(1 )-microglobulin reveals a potential haem-binding site. Biochem. J. 2012 Jul 15;445(2):175-82.

[0759] Olsson, M. G. et al., Free Radio Res, 2008, 42, p.725-36. Rutardottir, S. et al., Biochim Biophys Acta, 2016, 1864, p. 29-41.

[0760] Akerstrom B, Gram M. A1 M, an extravascular tissue cleaning and housekeeping protein. Free Radic Biol. Med. 2014 Sep;74:274-82.

[0761] Akerstrom, B. et al., J Biol Chem, 2007, 282, p. 31493-503.

[0762] Yang PY, Zou H, Chao E, Sherwood L, Nunez V, Keeney M, Ghartey-Tagoe E, Ding Z, Quirino H, Luo X, Welzel G, Chen G, Singh P, Woods AK, Schultz PG, Shen W. Engineering a long-acting, potent GLP-1 analog for microstructure-based transdermal delivery. Proc Natl Acad Sci U S A. 2016 Apr 12;113(15):4140-5. doi:

[0763] 10.1073 / pnas.1601653113. Epub 2016 Mar 28. PMID: 27035989; PMCID: PMC4839405.

[0764] Zhao F, Zhou Q, Cong Z, Hang K, Zou X, Zhang C, Chen Y, Dai A, Liang A, Ming Q, Wang M, Chen LN, Xu P, Chang R, Feng W, Xia T, Zhang Y, Wu B, Yang D, Zhao L, Xu HE, Wang MW. Structural insights into multiplexed pharmacological actions of tirzepatide and peptide 20 at the GIP, GLP-1 or glucagon receptors. Nat Commun. 2022 Feb 25;13(1 ):1057. doi: 10.1038 / S41467-022-28683-0. PMID: 35217653; PMCID: PMC8881610.

Claims

Claims1 . An agent comprising a polypeptide consisting of i. 12 to 80 amino acid residues, wherein the polypeptide comprises the amino acid sequence GSTCPWLKKIX1(SEQ ID NO: 20) wherein X1is M, K, R, or Nle, or ii. 30 to 80 amino acid residues, wherein the polypeptide comprises the amino acid sequence GATEAEISMTSTX2WRKGVCEETSGAYEKTD (SEQ ID NO: 21 ) wherein X2is H, R, or K.

2. An agent comprising a polypeptide consisting of 10 to 80 amino acid residues, wherein the polypeptide comprises a fragment of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 28, or SEQ ID NO: 29, wherein:X3is selected from D, N, or G;X4is selected from M, K, R, Nle;X5is selected from H, R, or K;X6is selected from D, N, or E; andX7is selected from M or Nle; said fragment being at least 10 amino acid residues in length, wherein the fragment comprises an amino acid residue corresponding to C34 or C72 of SEQ ID NO: 30.

3. An agent comprising a polypeptide consisting of at least 10 amino acid residues, wherein the polypeptide comprises a fragment of alpha-1 - microglobulin or a variant of alpha-1 -microglobulin, said fragment being at least 10 amino acid residues in length, wherein the fragment comprises an amino acid residue corresponding to C34 or C72 of SEQ ID NO: 30.

4. The agent according to any one of the preceding claims, wherein the polypeptide comprises the amino acid sequence GSTCPWLKKIX1(SEQ ID NO: 20) wherein X1is M, K, R, or Nle.

5. The agent according to any one of the preceding claims, wherein the polypeptide comprise the amino acid sequenceGATEAEISMTSTX2WRKGVCEETSGAYEKTD (SEQ ID NO: 21 ) wherein X2is H, R, or K.

6. The agent according to any one of the preceding claims, wherein the polypeptide comprises the amino acid sequence GSTCPWLKKIM (SEQ ID NO:22).

7. The agent according to any one of the preceding claims, wherein the polypeptide comprises the amino acid sequence GSTCPWLKKIK (SEQ ID NO:23).

8. The agent according to any one of the preceding claims, wherein the polypeptide comprises the amino acid sequence GSTCPWLKKIR (SEQ ID NO:24).

9. The agent according to any one of the preceding claims, wherein the polypeptide comprises the amino acid sequence GSTCPWLKKINIe (SEQ ID NO: 145).

10. The agent according to any one of the preceding claims, wherein the polypeptide comprises the amino acid sequence GATEAEISMTSTHWRKGVCEETSGAYEKTD (SEQ ID NO: 25).11 . The agent according to any one of the preceding claims, wherein the polypeptide comprises the amino acid sequence GATEAEISMTSTRWRKGVCEETSGAYEKTD (SEQ ID NO: 26).

12. The agent according to any one of the preceding claims, wherein the polypeptide comprises the amino acid sequence GATEAEISMTSTKWRKGVCEETSGAYEKTD (SEQ ID NO: 27).

13. The agent according to any one of the preceding claims, wherein the polypeptide comprises a fragment of any one of SEQ ID NO: 28 or SEQ ID NO:

14. The agent according to any one of the preceding claims, wherein the polypeptide comprises a fragment of any one of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19.

15. The agent according to any one of the preceding claims, wherein the polypeptide is a fragment of any one of SEQ ID NO: 28 or SEQ ID NO: 29.

16. The agent according to any one of the preceding claims, wherein the polypeptide is a fragment of any one of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19.

17. The agent according to any one of the preceding claims, wherein the polypeptide consists of at least 13 amino acid residues, such as at least 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, such as at least 24 amino acid residues.

18. The agent according to any one of the preceding claims, wherein the polypeptide consists of no more than 79 amino acid residues, such as no more than 78, 77, 76, 75, 74, 73, 72, 71 , 70, 69, 68, 67, 66, 65, 64, 63, 62, 61 , 60, 59, 58, 57, 56, such as no more than 55 amino acid residues.

19. The agent according to any one of the preceding claims, wherein the polypeptide comprises an amino acid residue corresponding to C34 or C72 of SEQ ID NO: 30.

20. The agent according to any one of the preceding claims, wherein the polypeptide comprises an amino acid residue corresponding to C34 of SEQ ID NO: 30.21 . The agent according to any one of the preceding claims, wherein the polypeptide comprises an amino acid residue corresponding to C72 of SEQ ID NO: 30.

22. The agent according to any one of the preceding claims, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of:i. DDDDKQVQENFDISRIYGKWYNLAIGSTCPWLKKIMDRMTVSTLVL GEGAT (SEQ ID NO: 1), ii. DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGAT (SEQ ID NO: 2), ill. DDKGPVPTPPDNIQVQENFDISRIYGKWYNLAIGSTCPWLKKIM (SEQ ID NO: 3), iv. KGSTCPWLKKIMDRMTVSTLVLGEGAT (SEQ ID NO: 4), v. GSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTHWRKGVCEETS GAYEK (SEQ ID NO: 6), vi. DDDDKGPVPTPPDNIQVQENFDISRIYGKWYNLAIGSTCPWLKKIMD RM (SEQ ID NO: 7), vii. GSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTHWRKGVCEET (SEQ ID NO: 8), viii. GSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTHWRK (SEQ ID NO: 9), ix. GSTCPWLKKIMDRMTVSTLVLGEGATEAEISMT (SEQ ID NO: 10), x. GSTCPWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 11 ), xi. NLAIGSTCPWLKKIMDR (SEQ ID NO: 12), xii. GEGATEAEISMTSTHWRKGVCEETSGAYEKTDTDG (SEQ ID NO: 13), xiii. GATEAEISMTSTHWRKGVCEETSGAYEKTD (SEQ ID NO: 14), xiv. GSTCPWLKKIMDRMTVSTLDLGEG (SEQ ID NO: 31 ), xv. GSTCPWLKKIMDRMTVSTLSLGEG (SEQ ID NO: 32), and xvi. GSTCPWLKKIMDRMTVSTLVLGEGATE (SEQ ID NO: 33).

23. The agent according to any one of the preceding claims, wherein the polypeptide is selected from the group consisting of: i. DDDDKQVQENFDISRIYGKWYNLAIGSTCPWLKKIMDRMTVSTLVL GEGAT (SEQ ID NO: 1), ii. DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGAT (SEQ ID NO: 2), ill. DDKGPVPTPPDNIQVQENFDISRIYGKWYNLAIGSTCPWLKKIM (SEQ ID NO: 3),iv. KGSTCPWLKKIMDRMTVSTLVLGEGAT (SEQ ID NO: 4), v. GSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTHWRKGVCEETS GAYEK (SEQ ID NO: 6), vi. DDDDKGPVPTPPDNIQVQENFDISRIYGKWYNLAIGSTCPWLKKIMD RM (SEQ ID NO: 7), vii. GSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTHWRKGVCEET (SEQ ID NO: 8), viii. GSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTHWRK (SEQ ID NO: 9), ix. GSTCPWLKKIMDRMTVSTLVLGEGATEAEISMT (SEQ ID NO: 10), x. GSTCPWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 1 1 ), xi. NLAIGSTCPWLKKIMDR (SEQ ID NO: 12), xii. GEGATEAEISMTSTHWRKGVCEETSGAYEKTDTDG (SEQ ID NO: 13), xiii. GATEAEISMTSTHWRKGVCEETSGAYEKTD (SEQ ID NO: 14), xiv. GSTCPWLKKIMDRMTVSTLDLGEG (SEQ ID NO: 31 ), xv. GSTCPWLKKIMDRMTVSTLSLGEG (SEQ ID NO: 32), and xvi. GSTCPWLKKIMDRMTVSTLVLGEGATE (SEQ ID NO: 33).

24. The agent according to any one of the preceding claims, wherein the polypeptide is effective in reducing cytochrome c.

25. The agent according to any one of the preceding claims, wherein the polypeptide has at least 35 % of the cytochrome c reduction activity of SEQ ID NO: 16, such as at least 36 %, 37 %, 38 %, 39 %, 40 %, 41 %, or 42 % of the cytochrome c reduction activity of SEQ ID NO: 16.

26. The agent according to any one of the preceding claims, wherein the polypeptide has an EC50 value for reduction of cytochrome c of at most 500 pM, such as at most 400 pM, 300 pM, 200 pM, 100 pM, or 50 pM.

27. The agent according to any one of the preceding claims, wherein the polypeptide binds heme.

28. The agent according to any one of the preceding claims, wherein the polypeptide is an antioxidant.

29. The agent according to any one of the preceding claims, wherein the agent further comprises a moiety capable of altering the biophysical properties of the polypeptide.

30. An agent comprising a biologically active polypeptide consisting of i. 12 to 80 amino acid residues, wherein the polypeptide comprises the amino acid sequence GSTCPWLKKIX1(SEQ ID NO: 20) wherein X1is M, K, R, or Nle, or ii. 30 to 80 amino acid residues, wherein the polypeptide comprises the amino acid sequence GATEAEISMTSTX2WRKGVCEETSGAYEKTD (SEQ ID NO: 21 ) wherein X2is H, R, or K.

31. An agent comprising a biologically active polypeptide consisting of 10 to 80 amino acid residues, wherein the polypeptide comprises a fragment of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 28, or SEQ ID NO: 29, wherein:X3is selected from D, N, or G;X4is selected from M, K, R, or Nle;X5is selected from H, R, or K;X6is selected from D, N, or E; andX7is selected from M or Nle; said fragment being at least 10 amino acid residues in length, and wherein the fragment comprises an amino acid residue corresponding to C34 or C72 of SEQ ID NO: 30.

32. An agent comprising a biologically active polypeptide consisting of at least 10 amino acid residues, wherein the polypeptide comprises a fragment of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 28, or SEQ ID NO: 29, wherein:X3is selected from D, N, or G;X4is selected from M, K, R, or Nle;X5is selected from H, R, or K;X6is selected from D, N, or E; andX7is selected from M or Nle; said fragment being at least 10 amino acid residues in length, and wherein the fragment comprises an amino acid residue corresponding to C34 or C72 of SEQ ID NO: 30.

34. An agent comprising a biologically active polypeptide having a length of from 10 to 92 amino acid residues, wherein the polypeptide comprises a fragment selected from position 1 to position 92 of human wt alpha-1 -microglobulin, A1 M, SEQ ID NO: 83:GPVPTPPDNI QVQENFNISR IYGKWYNLAI GSTCPWLKKI MDRMTVSTLV LGEGATEAEI SMTSTRWRKG VCEETSGAYE KTDTDGKFLY HK, wherein the fragment has a length of from 10 to 92 amino acid residues, and wherein the fragment includes one or more of Y22, C34, K69 or K92, where the positions refer to the positions in human wtA1 M (SEQ ID NO: 30).

35. The agent according to any one of the preceding claims, wherein the fragment is selected from position 1 to position 92 of human wtA1 M, and wherein the fragment has at least 50% identity with the corresponding fragment of human wtA1 M (SEQ ID NO: 30).

36. The agent according to any one of the preceding claims, wherein the fragment has a length of from 15 to 80 amino acid residues.

37. The agent according to any one of the preceding claims, wherein the fragment has a length of from 20 to 75 amino acid residues.

38. The agent according to any one of the preceding claims, wherein the fragment includes C34, wherein the position refers to the position in human wtA1 M (SEQ ID NO: 30).

39. The agent according to any one of the preceding claims, wherein the fragment includes Y22, wherein the position refers to the position in human wtA1 M (SEQ ID NO: 30).

40. The agent according to any one of the preceding claims, wherein the fragment includes K92, wherein the position refers to the position in human wtA1 M (SEQ ID NO: 30).41 . The agent according to any one of the preceding claims, wherein the fragment is selected from position 22 to position 92 of human wtA1 M (SEQ ID NO: 84):YGKWYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDGKFLYHK, wherein the fragment has a length of from 10 to 71 amino acid residues.

42. The agent according to any one of the preceding claims, wherein the fragment is selected from one of the following positions of human wt A1 M (SEQ ID NO: 30): i) Position 22 to position 62 (SEQ ID NO: 85), ii) Position 26 to position 62 (SEQ ID NO: 86), ill) Position 30 to position 62 (SEQ ID NO: 87), iv) position 31 to position 56 (SEQ ID NO: 88), v) position 26 to position 56 (SEQ ID NO: 89), vi) position 26 to position 92 (SEQ ID NO: 90), vii) position 30 to position 92 (SEQ ID NO: 91 ), or viii) position 31 to position 92 (SEQ ID NO: 92).

43. The agent according to any one of the preceding claims comprising an amino acid sequence having a sequence identity of 50% of more to one of the following sequences of human wt A1 M (SEQ ID NO: 30): i) Position 22 to position 62 (SEQ ID NO: 85), ii) Position 26 to position 62 (SEQ ID NO: 86), ill) Position 30 to position 62 (SEQ ID NO: 87), iv) position 31 to position 56 (SEQ ID NO: 88), v) position 26 to position 56 (SEQ ID NO: 89), vi) position 26 to position 92 (SEQ ID NO: 90), vii) position 30 to position 92 (SEQ ID NO: 91 ), viii) position 31 to position 92 (SEQ ID NO: 92).

44. The agent according to any one of the preceding claims comprising an amino acid sequence corresponding to one of the following sequences of human wt A1 M: i) Position 22 to position 62 (SEQ ID NO: 85), ii) Position 26 to position 62 (SEQ ID NO: 86), ill) Position 30 to position 62 (SEQ ID NO: 87), iv) position 31 to position 56 (SEQ ID NO: 88), v) position 26 to position 56 (SEQ ID NO: 89), vi) position 26 to position 92 (SEQ ID NO: 90), vii) position 30 to position 92 (SEQ ID NO: 91 ), viii) position 31 to position 92 (SEQ ID NO: 92).

45. The agent according to any one of the preceding claims, wherein the polypeptide at the N-terminal end and / or the C-terminal end comprises an amino acid sequence, Z, having a length of from 1 to 20 amino acid residues.

46. The agent according to any one of the preceding claims, wherein Z comprises one or more lysine, K, residue(s), one or more glutamic acid, E, residue(s), one or more arginine, R, residue(s) and / or one or more aspartic acid, D, residue(s).

47. The agent according to any one of the preceding claims, wherein Z comprises one or more hydrophobic amino acid residue(s).

48. The agent according to any one of the preceding claims, wherein Z comprises one or more of histidine H, residue(s), one or more of glutamine, G, residue(s), and / or one or more of isoleucine, I residue(s).

49. The agent according to any one of the preceding claims, comprising a sequence selected from:Y-rn -K, Y-F12-C, or C-F13-K, wherein fl 1 has at least 50% sequence identity with SEQ ID NO: 100, which is GKWYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEE TSGAYEKTDTDGKFLYH (SEQ ID NO: 100),F12 has at least 50% sequence identity with SEQ ID NO: 101 , which isGKWYNLAIGST (SEQ ID NO: 101 ), and FI3 has at least 50% sequence identity with SEQ ID NO: 102, which is PWLKKIMDRMTVSTLVLGEGATEAEISMTSTRWRKGVCEETSGAYEKTDTDG KFLYH (SEQ ID NO: 102).

50. The agent according to any one of the preceding claims having at least 50% sequence identity with SEQ ID NO: 83.

51. An agent having a length of from 10 to 100 amino acid residues, wherein the polypeptide comprises a fragment selected from position 1 to position 183 of human wt alpha-1 -microglobulin, A1 M, SEQ ID NO: 30, wherein the fragment has a length of from 10 to 100 amino acid residues, and wherein the fragment includes one or more of Y22, C34, K69, K92, K118, H122, Y132, L180, 1181 , P182 and R183.

52. The agent according to any one of the preceding claims, wherein the fragment is selected from position 1 to position 183 of human wtA1 M (SEQ ID NO: 30), and wherein the fragment has at least 50% identity with the corresponding fragment of human wtA1 M (SEQ ID NO: 30).

53. The agent according to any one of the preceding claims, wherein the fragment has a length of from 15 to 80 amino acid residues.

54. The agent according to any one of the preceding claims, wherein the fragment has a length of from 20 to 75 amino acid residues.

55. The agent according to any one of the preceding claims, wherein the fragment includes one or more of Y22, C34, K69, K92, K118, H122, and Y132.

56. The agent according to any one of the preceding claims, wherein the fragment includes one or more of K92, K118, H122, Y132, L180, 1181 , P182 and R183.

57. The agent according to any one of the preceding claims, wherein the fragment includes one or more of L180, 1181 , P182 and R183.

58. The agent according to any one of the preceding claims, wherein the fragment is selected from position 22 to position 183 of human wtA1 M (SEQ ID NO: 105)59. The agent according to any one of the preceding claims, wherein the fragment is selected from position 22 to position 160 of human wtA1 M (SEQ ID NO: 106)60. The agent according to any one of the preceding claims, wherein the fragment is selected from position 51 to position 155 of human wtA1 M (SEQ ID NO: 107)61 . The agent according to any one of the preceding claims, wherein the fragment is selected from position 59 to position 135 of human wtA1 M (SEQ ID NO: 108)62. The agent according to any one of the preceding claims, wherein the fragment is selected from position 92 to position 183 of human wtA1 M (SEQ ID NO: 109)63. The agent according to any one of the preceding claims, wherein the fragment is selected from position 92 to position 135 of human wtA1 M (SEQ ID NO: 1 10)64. The agent according to any one of the preceding claims having a length corresponding to from 10 to 75 amino acid residues such as from 10 to 60 amino acid residues.

65. The agent according to any one of the preceding claims, wherein the fragment is selected from one of the following ranges of human wtA1 M: i) Position 22 to position 135 (SEQ ID NO: 1 10), ii) Position 26 to position 135 (SEQ ID NO: 1 11 ), ill) Position 30 to position 135 (SEQ ID NO: 1 12), iv) Position 31 to position 135 (SEQ ID NO: 1 13), v) Position 61 to position 135 (SEQ ID NO: 1 14), vi) Position 66 to position 135 (SEQ ID NO: 1 15), vii) Position 87 to position 135 (SEQ ID NO: 1 16), viii) Position 92 to position 135 (SEQ ID NO: 1 17).

66. The agent according to any one of the preceding claims comprising an amino acid sequence having a sequence identity of 50% or more of one of the following sequences of human wtA1 M (SEQ ID NO: 30): i) Position 22 to position 135 (SEQ ID NO: 1 10), ii) Position 26 to position 135 (SEQ ID NO: 1 11 ), ill) Position 30 to position 135 (SEQ ID NO: 1 12), iv) Position 31 to position 135 (SEQ ID NO: 1 13), v) Position 59 to position 135 (SEQ ID NO: 108), vi) Position 66 to position 135 (SEQ ID NO: 1 15), vii) Position 87 to position 135 (SEQ ID NO: 1 16), viii) Position 92 to position 135 (SEQ ID NO: 1 17).

67. The agent according to any one of the preceding claims, comprising an amino acid sequence selected from one of the following sequences of human wtA1 M (SEQ ID NO: 30): i) Position 59 to position 135 (SEQ ID NO: 108), ii) Position 66 to position 135 (SEQ ID NO: 1 15), ill) Position 87 to position 135 (SEQ ID NO: 1 16), iv) Position 92 to position 135 (SEQ ID NO: 1 17).

68. The agent according to any of the preceding claims, wherein the polypeptide at the N- terminal end and / or the C-terminal end comprises an amino acid sequence, Z, having a length of from 1 to 20 amino acid residues.

69. The agent according to any one of the preceding claims, wherein Z comprises one or more lysine, K, residue(s), one or more glutamic acid, E, residue(s), one or more arginine, R, residue(s) and / or one or more aspartic acid, D, residue(s).

70. The agent according to any one of the preceding claims, wherein Z comprises one or more hydrophobic amino acid residue(s).71 . The agent according to any one of the preceding claims, wherein Z comprises one or more of histidine H, residue(s), one or more of glutamine, G, residue(s), and / or one or more of isoleucine, I, residue(s).

72. The agent according to any one of the preceding claims, wherein the polypeptide at the N-terminal end comprises Z, which is or comprises one or more of the following sequences: i) HHHHHHHHGGGGGIEGR (8H5GIEGR) (SEQ ID NO: 93), ii) HHHHHHHHDDDDK (8H4DK) (SEQ ID NO: 94), ill) HHHHHHDDDDK (6H4DK) (SEQ ID NO: 95), or iv) HHHHHHHH (8H) (SEQ ID NO: 96).

73. The agent according to any one of the preceding claims, wherein the polypeptide at the C-terminal end comprises Z, which is or comprises one or more of the following sequences: i) RGIEGGGGGHHHHHHHH (RGIE5G8H) (SEQ ID NO: 97) ii) KDDDDHHHHHHHH (K4D8H) (SEQ ID NO: 98) ill) KDDDDHHHHHH (K4D6H) (SEQ ID NO: 99) iv) HHHHHHHH (8H) (SEQ ID NO: 96)72. The agent according to any one of the preceding claims comprising an amino acid sequence having at least 50% sequence identity with SRIYGKWYNLAIGSTCPWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 103).

73. The agent according to any one of the preceding claims comprising an amino acid sequence having at least 50% sequence identity with ENFNISRIYGKWYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATEAE (SEQ ID NO: 104).

74. The agent according to any one of the preceding claims, wherein the polypeptide comprises one or more further modifications.

75. The agent according to any one of the preceding claims, wherein the further modification is a non-peptide modification.

76. The agent according to any one of the preceding claims, wherein the further modification is a replacement of one or more atoms or of one or more moieties.

77. The agent according to any one of the preceding claims, wherein the further modification is a peptide-based modification.

78. The agent according to any one of the preceding claims, wherein the polypeptide is further modified on the N-terminal end.

79. The agent according to any one of the preceding claims, wherein the polypeptide is further modified on the C-terminal end.

80. The agent according to any one of the preceding claims, wherein the polypeptide is further modified on a side chain.81 . The agent according to any one of the preceding claims, wherein the polypeptide is acetylated N-terminally.

82. The agent according to any one of the preceding claims, wherein the polypeptide is amidated C-terminally.

83. The agent according to any one of the preceding claims, wherein the polypeptide comprises a substitution with a non-natural amino acid.

84. The agent according to any one of the preceding claims, wherein the polypeptide comprises a substitution with a D-amino acid.

85. The agent according to any one of the preceding claims, wherein a residue in the polypeptide is replaced with the same amino acid in D-configuration.

86. The agent according to any one of the preceding claims, wherein the polypeptide comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions with D-amino acids.

87. The agent according to any one of the preceding claims, wherein the polypeptide comprises a sequence selected from the group consisting of: a. NLAIGSTCP[D-Trp]LKKIMDR (SEQ ID NO: 66), b. NLAIGSTCPW[D-Leu]KKIMDR (SEQ ID NO: 67), c. NLAIGSTCPWLK[D-Lys]IMDR (SEQ ID NO: 68), d. GSTCPWLK[D-Lys]IMDRMTVSTLVLGEG (SEQ ID NO: 69), e. GSTCPWLKKIMD[D-Arg]MTVSTLVLGEG (SEQ ID NO: 70), f. GSTCPWLKKIMDRMTVSTLV[D-Leu]GEG (SEQ ID NO: 71 ), g. GSTCPWLKKIMDRMTVSTL[D-Asp]LGEG (SEQ ID NO: 72), h. G[D-Ser]STCPWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 81 ), and i. [D-Asn]LAIGSTCPWLKKIMDR (SEQ ID NO: 82), or a sequence having at least 70 % sequence identity thereto, such as at least 75 %, at least 80 %, at least 85 %, at least 90 %, such as at least 95 % sequence identity thereto.

88. The agent according to any one of the preceding claims, wherein the polypeptide is selected from the group consisting of: a. NLAIGSTCP[D-Trp]LKKIMDR (SEQ ID NO: 66), b. NLAIGSTCPW[D-Leu]KKIMDR (SEQ ID NO: 67), c. NLAIGSTCPWLK[D-Lys]IMDR (SEQ ID NO: 68), d. GSTCPWLK[D-Lys]IMDRMTVSTLVLGEG (SEQ ID NO: 69), e. GSTCPWLKKIMD[D-Arg]MTVSTLVLGEG (SEQ ID NO: 70), f. GSTCPWLKKIMDRMTVSTLV[D-Leu]GEG (SEQ ID NO: 71 ), g. GSTCPWLKKIMDRMTVSTL[D-Asp]LGEG (SEQ ID NO: 72), h. G[D-Ser]STCPWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 81 ), and i. [D-Asn]LAIGSTCPWLKKIMDR (SEQ ID NO: 82).

89. The agent according to any one of the preceding claims, wherein one, two, or more methionine residues of the polypeptide have been substituted with norleucine.

90. The agent according to any one of the preceding claims, wherein the methionine residue corresponding to M41 of A1 M has been substituted with norleucine in the polypeptide.91 . The agent according to any one of the preceding claims, wherein the methionine residue corresponding to M44 of A1 M has been substituted with norleucine in the polypeptide.

92. The agent according to any one of the preceding claims, wherein both the methionine residues corresponding to M41 and M44 of A1 M has been substituted with norleucine in the polypeptide.

93. The agent according to any one of the preceding claims wherein the polypeptide is selected from the group consisting of: i. GSTCPWLKKI[Nle]DR[Nle]TVSTLVLGEG (SEQ ID NO:139), ii. GSTCPWLKKI[Nle]DR[Nle]TVSTL[D-Asp]LGEG (SEQ ID NO:140), ill. GSTCPWLKKI[Nle]DR[Nle]TVSTLVLGEG[D-Ala][D-Thr][D-Glu] (SEQ ID NO:141), iv. GSTCPWLKKI[Nle]DR[Nle]TKSTLVLGEG (SEQ ID NO:142), v. EEKYNLAIGSTCPWLKKI[Nle]DR[Nle]TVSTLVLGE (SEQ ID NO:143), and vi. GSTCPWLKKI[Nle]DR[Nle]TVSTL[D-Asp]LGEG (SEQ ID NO:144).

94. The agent according to any one of the preceding claims, wherein the polypeptide is PEGylated.

95. The agent according to any one of the preceding claims, wherein the PEGylation is at the N-terminal end, the C-terminal end, or on a side-chain.

96. The agent according to any one of the preceding claims, wherein the PEGylation is at the C-terminal end.

97. The agent according to any one of the preceding claims, wherein the PEGylation is with a PEG having a molecular weight of at most 60 kDa, such as at most 50 kDa, such as at most 40 kDa, such as at most 30 kDa, such as at most 20 kDa, such as at most 15 kDa, such as at most 10 kDa, such as at most 5 kDa.

98. The agent according to any one of the preceding claims, wherein the PEGylation is with any one of PEG1 to PEG50.

99. The agent according to any one of the preceding claims, wherein the PEGylation is with any one of PEG1 to PEG30.

100. The agent according to any one of the preceding claims, wherein the PEGylation is with any one of PEG2 to PEG20.101 . The agent according to any one of the preceding claims, wherein the PEGylation is with PEG2, PEG5, PEG10, or PEG20.

102. The agent according to any one of the preceding claims, wherein the PEGylated polypeptide comprises a structure selected from the group consisting of: a. GSTCPWLKKIMDRMTVSTLVLGEG-PEG2-CONH2 (SEQ ID NO: 34), b. GSTCPWLKKIMDRMTVSTLDLGEG-PEG2-CONH2 (SEQ ID NO: 35), c. AC-NLAIGSTCPWLKKIMDR-CONH2-PEG2 (SEQ ID NO: 39), d. AC-DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGAT-PEG2 (SEQ ID NO: 40), e. NLAIGSTCPWLKKIMDR-PEG5 (SEQ ID NO: 60), f. NLAIGSTCPWLKKIMDR-PEG10 (SEQ ID NO: 61 ), g. GSTCPWLKKIMDRMTVSTLVLGEG-PEG5 (SEQ ID NO: 62), h. GSTCPWLKKIMDRMTVSTLVLGEG-PEG10 (SEQ ID NO: 63), i. NLAIGSTCPWLKKIMDR-PEG20 (SEQ ID NO: 64), and j. GSTCPWLKKIMDRMTVSTLVLGEG-PEG20 (SEQ ID NO: 65),or a sequence having at least 70 % sequence identity thereto, such as at least 75 %, at least 80 %, at least 85 %, at least 90 %, such as at least 95 % sequence identity thereto.

103. The agent according to any one of the preceding claims, wherein the PEGylated polypeptide is selected from the group consisting of: a. GSTCPWLKKIMDRMTVSTLVLGEG-PEG2-CONH2(SEQ ID NO: 34) b. GSTCPWLKKIMDRMTVSTLDLGEG-PEG2-CONH2 (SEQ ID NO: 35) c. AC-NLAIGSTCPWLKKIMDR-CONH2-PEG2 (SEQ ID NO: 39) d. AC-DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGAT-PEG2 (SEQ ID NO: 40) e. NLAIGSTCPWLKKIMDR-PEG5 (SEQ ID NO: 60) f. NLAIGSTCPWLKKIMDR-PEG10 (SEQ ID NO: 61 ) g. GSTCPWLKKIMDRMTVSTLVLGEG-PEG5 (SEQ ID NO: 62) h. GSTCPWLKKIMDRMTVSTLVLGEG-PEG10 (SEQ ID NO: 63) i. NLAIGSTCPWLKKIMDR-PEG20 (SEQ ID NO: 64) j. GSTCPWLKKIMDRMTVSTLVLGEG-PEG20 (SEQ ID NO: 65)104. The agent according to any one of the preceding claims, wherein the polypeptide further comprises an albumin binding domain or a fragment of an albumin binding domain.

105. The agent according to any one of the preceding claims, wherein the albumin binding domain has the amino acid sequence of SEQ ID NO: 1 18.

106. The agent according to any one of the preceding claims, wherein the albumin binding domain fragment consists of 10 to 50 amino acid residues.

107. The agent according to any one of the preceding claims, wherein the albumin binding domain or fragment thereof is conjugated to the N-terminal end of the polypeptide, the C-terminal end of the polypeptide, or a side chain of the peptide.

108. The agent according to any one of the preceding claims, wherein the albumin binding domain or fragment thereof is conjugated to the polypeptide via a linking moiety.

109. The agent according to any one of the preceding claims, wherein the linker is a PEG linker.

110. The agent according to any one of the preceding claims, wherein the linking moiety is a peptide linker.

111. The agent according to any one of the preceding claims, wherein the linking moiety is selected from the group consisting of: a. GGGGGSAS (SEQ ID NO: 121), and b. GGGGSGGGGSGGGGSAS (SEQ ID NO: 122).

112. The agent according to any one of the preceding claims, wherein the albumin domain binding fragment comprises or consists of the amino acid sequence LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 118) or SDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 119).

113. The agent according to any one of the preceding claims, wherein the polypeptide is selected from the group consisting of: a. GSTCPWLKKIMDRMTVSTLVLGEGSDFYKRLINKAKTVEGVEALKLHIL AALP (SEQ ID NO: 36), b. GSTCPWLKKIMDRMTVSTLVLGEGGGGGSASSDFYKRLINKAKTVEG VEALKLHILAALP (SEQ ID NO: 37), c. GSTCPWLKKIMDRMTVSTLVLGEGGGGGSGGGGSGGGGSASSDFY KRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 38), d. NLAIGSTCPWLKKIMDRGGGGSASSDFYKRLINKAKTVEGVEALKLHIL AALP (SEQ ID NO: 41), e. NLAIGSTCPWLKKIMDRGGGGSGGGGSGGGGSASSDFYKRLINKAKT VEG VEALKLHILAALP (SEQ ID NO: 42),f. DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATGGGGSASSDFY KRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 43), g. DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATGGGGSGGGGS GGGGSASSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 44), and h. EEKYNLAIGSTCPWLKKIMDRMTESTLVLGEGGGGGSASLAEAKVLAN RELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 163). or a sequence having at least 70 % sequence identity thereto, such as at least 75 %, at least 80 %, at least 85 %, at least 90 %, such as at least 95 % sequence identity thereto.

114. The agent according to any one of the preceding claims, wherein the polypeptide is selected from the group consisting of: a. GSTCPWLKKIMDRMTVSTLVLGEGSDFYKRLINKAKTVEGVEALKLHIL AALP (SEQ ID NO: 36), b. GSTCPWLKKIMDRMTVSTLVLGEGGGGGSASSDFYKRLINKAKTVEG VEALKLHILAALP (SEQ ID NO: 37), c. GSTCPWLKKIMDRMTVSTLVLGEGGGGGSGGGGSGGGGSASSDFY KRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 38), d. NLAIGSTCPWLKKIMDRGGGGSASSDFYKRLINKAKTVEGVEALKLHIL AALP (SEQ ID NO: 41), e. NLAIGSTCPWLKKIMDRGGGGSGGGGSGGGGSASSDFYKRLINKAKT VEG VEALKLHILAALP (SEQ ID NO: 42), f. DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATGGGGSASSDFY KRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 43), and g. DDDDKYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATGGGGSGGGGS GGGGSASSDFYKRLINKAKTVEGVEALKLHILAALP (SEQ ID NO: 44).

115. The agent according to any one of the preceding claims, wherein the polypeptide comprises a modification of one or more amino acid residues.

116. The agent according to any one of the preceding claims, wherein the modification is selected from the group consisting of methylation such as N- methylation, dimethylation such as N-dimethylation or C-dimethylation, sidechain methylation, oxidation such as oxidation of cysteine or methionine, and dimerization, such as dimerization between two cysteine moieties.

117. The agent according to any one of the preceding claims, wherein the modification is cysteine carbamidomethylation (cysteine CAM).

118. The agent according to any one of the preceding claims, wherein the polypeptide comprises or consists of the amino acid sequence GSTC(Cam)PWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 48), wherein Cam specifies carbamidomethylation.

119. The agent according to any one of the preceding claims, wherein the polypeptide comprises homocysteine.

120. The agent according to any one of the preceding claims, wherein a cysteine moiety of the polypeptide is substituted with homocysteine.

121. The agent according to any one of the preceding claims, wherein the polypeptide comprises or consists of the amino acid sequence GST{hC}PWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 49), wherein hC is homocysteine.

122. The agent according to any one of the preceding claims, wherein the polypeptide comprises a penicillamine residue.

123. The agent according to any one of the preceding claims, wherein a cysteine moiety of the polypeptide is substituted with penicillamine.

124. The agent according to any one of the preceding claims, wherein the polypeptide comprises or consists of the amino acid sequence GST{Penc}PWLKKIMDRMTVSTLVLGEG (SEQ ID NO: 50), wherein Penc is a penicillamine residue.

125. The agent according to any one of the preceding claims, wherein the polypeptide comprises one or more methionine sulfoxides.

126. The agent according to any one of the preceding claims, wherein the polypeptide comprises or consists of the amino acid sequence GSTCPWLKKIM(ox)DRM(ox)TVSTLVLGEG (SEQ ID NO: 51 ), wherein M(ox) specifies methionine sulfoxide.

127. The agent according to any one of the preceding claims, wherein the polypeptide is provided as a TFA salt or a chloride salt.

128. The agent according to any one of the preceding claims, wherein the polypeptide is methylated or dimethylated.

129. The agent according to any one of the preceding claims, wherein the polypeptide comprises one or more of a modification selected from the group consisting of: a. arginine side chain methylation, b. lysine methylation, c. lysine dimethylation, d. threonine N-methylation, and e. isoleucine N-methylation.

130. The agent according to any one of the preceding claims, wherein the polypeptide comprises or consists of an amino acid sequence selected from the group consisting of: a. GSTCPWLKKIMD[Arg methylated side chain]MTVSTLVLGEG (SEQ ID NO: 73), b. GSTCPWLK[Lys di-methylated]IMDRMTVSTLVLGEG (SEQ ID NO: 74), c. GSTCPWLKKIMDRM[Thr N-Methylated]VSTLVLGEG (SEQ ID NO: 75), d. GSTCPWLKK[lle N-methylated]MDRMTVSTLVLGEG (SEQ ID NO: 76),e. NLAIGSTCPWLK[Lys di-methylated]IMDR (SEQ ID NO: 77), f. NLAIGS[Thr N-Methylated]CPWLKKIMDR (SEQ ID NO: 78), g. NLA[lle N-methylated]GSTCPWLKKIMDR (SEQ ID NO: 79), and h. NLAIGSTCPWLKKIMD[Arg methylated side chain] (SEQ ID NO: 80), or a sequence having at least 70 % sequence identity thereto, such as at least 75 %, at least 80 %, at least 85 %, at least 90 %, such as at least 95 % sequence identity thereto.131 . The agent according to any one of the preceding claims, wherein the polypeptide is cyclised, such as cyclised N-terminal to C-terminal.

132. The agent according to any one of the preceding claims, wherein the polypeptide comprises a linker moiety linking two amino acid residues of the polypeptide.

133. The agent according to any one of the preceding claims, wherein the linking moiety has the structure of formula (I):wherein R may be 1 , 2, 3, or 4 substituents independently selected from the group consisting of H, F, Cl, Br, I, NO2, NO, SH, SOH, SO2H, SO3H, alkyl, alkenyl, alkynyl, COOH, COOR2, CHO, OH, R2, and OR2, wherein R2 is alkyl, alkenyl, or alkynyl, wherein the dashed lines specify attachment points to the amino acid residues of the polypeptide.

134. The agent according to any one of the preceding claims, wherein the linking moiety has the structure of formula (la):formula (la),wherein the dashed lines specify attachment points to the amino acid residues of the polypeptide.

135. The agent according to any one of the preceding claims, wherein the polypeptide comprises a linker moiety linking two amino acid residues was prepared using 1 ,3-bis(bromomethyl)benzene.

136. The agent according to any one of the preceding claims, wherein the two linked amino acid residues are linked via groups independently selected from an N-terminal amine, a side chain heteroatom selected from S, N, and O, a C- terminal acid, and a C-terminal amide.

137. The agent according to any one of the preceding claims, wherein the polypeptide comprises or consists of an structure selected from the group consisting of: a. C1 -GSTCPWLKKIMDR-C1 -TVSTLVLGEG (SEQ ID NO: 54), b. C1 -NLAIGSTCPWLKKIMDRMTVS-C1 (SEQ ID NO: 55), c. C1 -AIGSTCPWLKKIMDRMT-C1 (SEQ ID NO: 56), and d. C1 -NLAIGSTCPWLKKIMDR-C1 (SEQ ID NO: 59), or a sequence having at least 70 % sequence identity thereto, such as at least 75 %, at least 80 %, at least 85 %, at least 90 %, such as at least 95 % sequence identity thereto, wherein C1 specifies the point of attachment of the linker moiety.

138. The agent according to any one of the preceding claims, wherein the polypeptide is selected from the group consisting of: a. C1 -GSTCPWLKKIMDR-C1 -TVSTLVLGEG (SEQ ID NO: 54), b. C1 -NLAIGSTCPWLKKIMDRMTVS-C1 (SEQ ID NO: 55), c. C1 -AIGSTCPWLKKIMDRMT-C1 (SEQ ID NO: 56), and d. C1 -NLAIGSTCPWLKKIMDR-C1 (SEQ ID NO: 59), wherein C1 specifies the point of attachment of the linker moiety.

139. The agent according to any one of the preceding claims, wherein the polypeptide is selected from the group consisting of: a. Cyclo(CIAc-EEKYNLAIGSTCPWLKKI[Nle]DR[Nle]TKSTLVLGE-Cys)- NH2 (SEQ ID NO: 160), and b. Cyclo(CIAc-EEKYNLAIGSTCPWLKKIMDRMTESTLVLGE-Cys)-NH2 (SEQ ID NO: 162).

140. The agent according to any one of the preceding claims, wherein the agent comprises a second polypeptide.141 . The agent according to any one of the preceding claims, wherein the second polypeptide is not a fragment of A1 M (SEQ ID NO: 120).

142. The agent according to any one of the preceding claims, wherein the agent comprises at most 80 contiguous amino acid residues of human A1 M (SEQ ID NO: 120) or at most 80 contiguous amino acid residues of a sequence having at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, or at least 95 % sequence identity to human A1 M (SEQ ID NO: 120).

143. The agent according to any one of the preceding claims, wherein the second polypeptide is albumin binding domain (ABD1 , SEQ ID NO: 118).

144. The agent according to any one of the preceding claims, wherein the second polypeptide is albumin binding domain fragment (SEQ ID NO: 119).

145. The agent according to any one of the preceding claims, wherein the second polypeptide is a Human Fc fragment, such as a Human IgG Fc fragment, such as a Human IgG 1 Fc fragment, such as a Human IgG 1 Fc fragment having SEQ ID NO: 138.

146. The agent according to any one of the preceding claims, wherein the polypeptide is selected from the group consisting of:a. GSTCPWLKKIMDRMTKSTL[d-D]LGEG (SEQ ID NO:146) b. EEKYNLAIGSTCPWLKKIMDRMTESTLDLGE (SEQ ID NO:147) c. EEKYNLAKGSTCPWLKKIMDRMTESTLDLGE (SEQ ID NO:148) d. GSTCPWLKKI[NLe]DR[NLe]TKSTL[d-D]LGEG (SEQ ID NO:149) e. EEKYNLAIGSTCPWLKKI[NLe]DR[NLe]TESTLDLGE (SEQ ID NQ:150) f. EEKYNLAKGSTCPWLKKI[NLe]DR[NLe]TESTLDLGE (SEQ ID NO:151 ) g. GSTCPWLKKI[NLe]DR[NLe]TK[N-Meth S]TL[D]LGEG (SEQ IDNO:152) h. EEKYNLAIGSTCPWLKKI[NLe]DR[NLe]TE[N-Meth S]TLDLGE (SEQ ID NO:153)I. EEKYNLAKGSTCPWLKKI[NLe]DR[NLe]TE[N-Meth S]TLDLGE (SEQ ID NO: 154) j. Ac-EEKYNLAIGSTCPWLKKI[Nle]DR[Nle]TKSTLVLGE-NH2 (SEQ ID NO:155) k. Ac-EEKYNLAIGSTCPWLKKI[Nle]DR[Nle]TESTLVLGE-NH2 (SEQ ID NO: 161)147. The agent according to any one of the preceding claims, wherein the polypeptide further comprises a fatty acid.

148. The agent according to any one of the preceding claims, wherein the polypeptide is conjugated to a fatty acid.

149. The agent according to any one of the preceding claims, wherein the fatty acid is conjugated to the N-terminal end of the polypeptide, the C-terminal end of the polypeptide, or a side chain of the peptide.

150. The agent according to any one of the preceding claims, wherein the fatty acid is conjugated to the polypeptide via a linking moiety.151 . The agent according to any one of the preceding claims, wherein the linking moiety is yGlu-2xQEG.

152. The agent according to any one of the preceding claims, wherein the linking moiety is OEG.

153. The agent according to any one of the preceding claims, wherein the fatty acid is a C20 fatty diacid moiety (C20DA).

154. The agent according to any one of the preceding claims, wherein the polypeptide in conjugated to yGlu-2xOEG-C20 diacid.

155. The agent according to any one of the preceding claims, wherein the polypeptide is selected from the group consisting of: a. Ac-EEKYNLAIGSTCPWLKKI[Nle]DR[Nle]T[K([C20DA]-[yGlu]-[QEG]- [OEG]-)]STLVLGE-NH2 (SEQ ID NO:156 & SEQ ID NO:164) b. [C20DA]-[yGlu]-[OEG]-[OEG]- EEKYNLAIGSTCPWLKKI[Nle]DR[Nle]TKSTLVLGE-NH2 (SEQ ID NO:157) c. Ac-GSTCPWLK[K([C20DA]-[yGlu]-[OEG]-[OEG]- )]l[NLe]DR[NLe]TK[NMeSer]TL[D]LGEG-NH2 (SEQ ID NO:158 & SEQ ID NO:165) d. [C20DA]-[yGlu]-[QEG]-[QEG]- GSTCPWLKKI[NLe]DR[NLe]TK[NMeSer]TL[D]LGEG-NH2 (SEQ ID NO:159)156. A polynucleotide encoding the polypeptide or the agent according to any one of the preceding claims.

157. A vector comprising the polynucleotide according to any one of the preceding claims.

158. A cell comprising the polynucleotide according to any one of the preceding claims or the vector accordingly to any one of the preceding claims.

159. A pharmaceutical composition comprising the agent according to any one of the preceding claims, and at least one pharmaceutically acceptable carrier, excipient, or diluent.

160. The pharmaceutical composition according to any one of the preceding claims, wherein the composition is formulated for oral administration.161 . The agent according to any one of the preceding claims for use in medicine.

162. An agent as defined in any one of the preceding claims for use in the treatment of cerebral intraventricular hemorrhage (IVH).

163. The agent according to any one of the preceding claims, for use in the treatment of a kidney disease, disorder or injury.

164. The agent for use according to any one of the preceding claims, wherein the kidney disease, disorder or injury is induced by, propagated by and / or associated with oxidative stress.

165. The agent for use according to any one of the preceding claims, wherein the oxidative stress is induced by, propagated by, or associated with heme and / or heme-related products.

166. The agent for use according to any one of the preceding claims, wherein the oxidative stress is induced by, propagated by, or associated with cell-free heme.

167. The agent for use according to any one of the preceding claims, wherein the kidney disease, disorder or injury presents with haematuria.

168. The agent for use according to any one of the preceding claims, wherein the kidney disease, disorder or injury presents with glomerular haematuria.

169. The agent for use according to any one of the preceding claims, wherein the kidney disease, disorder or injury is caused by, induced by and / or propagated by glomerulonephritis.

170. The agent for use according to any one of the preceding claims, wherein the kidney disease, disorder or injury is caused by, induced by and / or propagated by nephrotoxicity.171 . The agent for use according to any one of the preceding claims, wherein the kidney disease, disorder or injury is acute kidney injury (AKI) .

172. The agent for use according to any one of the preceding claims, wherein the acute kidney injury (AKI) is ischemia-reperfusion injury (IRI).

173. The agent for use according to any one of the preceding claims, wherein the acute kidney injury (AKI) is caused by, induced by and / or propagated by sepsis.

174. The agent for use according to any one of the preceding claims, wherein the acute kidney injury (AKI) is caused by, induced by and / or propagated by cisplatin.

175. The agent for use according to any one of the preceding claims, wherein the kidney disease, disorder or injury is acute kidney disease (AKD).

176. The agent for use according to any one of the preceding claims, wherein the kidney disease, disorder or injury is chronic kidney disease (CKD).

177. The agent for use according to any one of the preceding claims, wherein the CKD is associated with and / or presents with haematuria.

178. The agent for use according to any one of the preceding claims, wherein the CKD is selected from the group consisting of IgA Nephropathy, primary focal segmental glomerulosclerosis (FSGS), Alport syndrome, thin basement membrane disease (TBMN), C3 glomerulonephritis (C3GN), Lupus nephritis, ANCA-associated vasculitis, ANCA-associated glomerulonephritis, diabetic kidney disease (DKD), hypertensive nephrosclerosis, polycystic kidney disease, non-diabetic CKD, non-proteinuric CKD, interstitial nephritis, drug-induced CKD, CKD based on an autoimmune disease, CKD of unknown / unspecific causes,and CKD based on genetic abnormalities including genetic abnormalities of APOL1 nephropathy.

179. The agent for use according to any one of the preceding claims, wherein the CKD is caused by glomerulonephritis.

180. The agent for use according to any one of the preceding claims, wherein the CKD is selected from the group consisting of IgA Nephropathy, primary focal segmental glomerulosclerosis (FSGS), Alport syndrome, thin basement membrane disease (TBMN), C3 glomerulonephritis (C3GN), Lupus nephritis, ANCA-associated vasculitis, or ANCA-associated glomerulonephritis.181 . The agent according to any one of the preceding claims, for use in the treatment of a disease or disorder selected from the group consisting of a disease or disorder caused by or associated with haemorrhage, haemolysis, or anaemia; a systemic haem disease; sickle cell anaemia; haemolytic anaemia; diamond blackfan anaemia; malaria; sepsis; vascular injury; peripheral artery disease; a vascular disease or disorder such as a cardiovascular disease or disorder; atherosclerosis; ischemic heart disease; thrombotic vascular disease; heart failure; myocardial infarction; a CNS disease or disorder such as haemorrhagic stroke or ischemic stroke; and tissue or nerve injury due to haemolytic trauma or due to haemorrhage.

182. A method of treating a disease or disorder comprising administering a therapeutically effective amount of the agent according to any one of the preceding claims.

183. A method for reducing oxidative stress in a subject, said method comprising administering the agent according to any one of the preceding claims to said subject.

184. A method of reducing oxidative stress in a cell, said method comprising contacting said cell with the agent according to any one of the preceding claims.

185. Use of the agent according to any one of the preceding claims in the manufacture of a medicament for treatment of a disease or disorder.