Arginase-insulin fusion protein

Abstract

The present invention discloses a fusion protein of insulin and arginase, which is useful as an antitumor drug, an antiobesity drug, or a type 2 diabetes drug.

Description

[Technical field]

[0001] The present invention relates to fusion proteins of arginase and insulin and to their use in medicine, in particular for the treatment of cancer and metabolic disorders such as obesity or diabetes, e.g. type 2 diabetes. [Background technology]

[0002] Arginine depletion has been shown to be useful in the treatment of some cancers, such as hepatocellular carcinoma and melanoma, and possibly many other cancers based on in vitro studies. The use of arginine depleting enzymes, such as arginase, in cancer treatment has been described, for example, by Shen et al. (Cell Death & Disease 8 (2017), e2720), Zou et al. (Biomedicine & Pharmacotherapy 118 (2019), 109210), Al-Koussa et al. (Cancer Cell International 20 (2020) Article number 150), and Zhang et al. (Cancer Letters 502 (2012), 58-70), the contents of which are incorporated herein by reference.

[0003] Our own studies have shown that the use of arginine-converting enzymes is essential but not sufficient to cause and maintain the deep systemic arginine depletion necessary to rapidly and selectively kill cancer cells.

[0004] The use of an insulin / glucose clamp in parallel with the enzymatic degradation of arginine makes the challenge of deep arginine depletion more tractable. Insulin is a growth factor and therefore promotes protein synthesis and inhibits protein degradation. This is crucial when the challenge is the removal of any amino acid from the circulation, particularly arginine, a semi-essential amino acid that is under tight homeostatic control.

[0005] The increase in vascular permeability by insulin also aids in the uptake of therapeutic enzymes into the interstitial fluid spaces close to where most cancer cells reside.

[0006] And finally, insulin may also play a role in delivering arginine-degrading enzymes into cancer cells by stimulating endocytosis. For this to work, the enzyme molecule must be in close proximity when the insulin molecule attaches to the insulin receptor, a process governed by chance and enzyme concentration.

[0007] It was an object of the present invention to overcome the drawbacks associated with previous treatment schedules involving amino acid depletion, such as arginine depletion. Summary of the Invention

[0008] A first aspect of the present invention provides a fusion protein comprising a first domain and a second domain, The first domain comprises an amino acid degrading enzyme, and the second domain comprises insulin. Related to fusion proteins.

[0009] A further aspect of the invention relates to a nucleic acid molecule encoding said fusion protein.

[0010] A further aspect of the invention relates to a host cell transfected with said nucleic acid molecule.

[0011] A further aspect of the invention is a method for producing said fusion protein, comprising the steps of: by culturing said host cell and obtaining said fusion protein from said host cell or from the culture medium; Related to the method.

[0012] A further aspect of the invention relates to said fusion protein for use in medicine.

[0013] In one embodiment, the first domain (enzyme) is located N-terminal to the second domain (insulin). In a further embodiment, the second domain (insulin) is located N-terminal to the first domain (enzyme).

[0014] In certain embodiments, a fusion protein is a genetic fusion, which can be produced in a recombinant host cell by expression of a nucleic acid molecule, particularly a DNA molecule encoding the fusion protein or a precursor thereof, and optional subsequent processing.

[0015] In one embodiment, the fusion protein is a non-genetic fusion, where the first domain and the second domain are produced separately, eg, in a recombinant host cell, and then linked to each other, eg, by a covalent bond.

[0016] The first domain of the fusion protein comprises amino acid decomposition enzyme.In some embodiments, the amino acid decomposition enzyme is arginine decomposition enzyme, such as arginine deiminase (ADI; EC 3.5.3.6; UniProt-P23793) or arginase.In a specific embodiment, the amino acid decomposition enzyme is human liver arginase (human arginase-1; ARG1; EC 3.5.3.1; Uni-Prot-P05089) or human kidney arginase (human arginase-2; ARG2; EC 3.5.3.1; Uni-Prot-P78540).

[0017] Modifications of human liver arginase (ARG1) or human kidney arginase (ARG2) to replace manganese with cobalt and shift the pH optimum to that of plasma are also particularly suitable for fusion with insulin according to the invention. 2+ Modified recombinant human arginase I is Stone EM, Glazer ES, Chantranupong L et al. (Replacing Mn(2+) with Co(2+) in human arginase enhances cytotoxicity toward L-arginine auxotrophic cancer cell lines, ACS Chem Biol. 2010;5(3):333-342, doi:10.1021 / cb900267j) and in US 20121 / 0189371 A1, the contents of which are incorporated herein by reference.

[0018] Several other amino acids have been targeted for cancer therapy, for example tryptophan by tryptophan dioxygenase (TDO2; EC 1.13.11.11; UniProt-P48775) or methionine by S-adenosylmethionine synthase (MAT1A; EC 2.5.1.6; UniProt-Q00266). However, arginine depletion is considered the most effective approach for cancer therapy.

[0019] Since the early 70s, asparaginase has been the most successfully used enzyme therapy for cancer, specifically for childhood acute lymphoblastic leukemia (ALL). Asparaginase is only active in the form of a tetramer, which is too large, about 130 kDa, to be used as such. According to the present invention, it is delivered in a dissociated form, for example dissolved in urea in the form of a monomer, where each of the monomers is fused to insulin, as described in WO2020 / 245041, the contents of which are incorporated herein by reference. In such a case, extravasation is possible, followed by reconstitution in the interstitial fluid into a tetramer, resulting in the active form of the enzyme. Thus, asparaginase is also a preferred enzyme for use in the present invention.

[0020] In certain embodiments, the amino acid degrading enzyme is a monomeric protein, such as a monomeric arginase.

[0021] The second domain of the fusion protein comprises insulin, including its precursors, such as proinsulin (from which insulin can be obtained by enzymatic cleavage, including autocleavage).

[0022] In one embodiment, the insulin is human insulin or an insulin analogue, such as a fast acting insulin, such as insulin glulisine, insulin aspart, insulin lispro, or a slow acting insulin, such as NPH insulin, insulin glargine, insulin detemir, or insulin degludec. These insulins typically comprise an A chain and a B chain linked by an S-S bridge, and can be obtained by cleavage from the corresponding proinsulin.

[0023] In one embodiment, the fusion proteins of the invention are produced as precursors in which the first domain comprises proinsulin (which is subsequently cleaved, eg, by autocatalysis, to the corresponding insulin).

[0024] Alternatively, the insulin may be a single-chain insulin, for example an insulin or insulin analogue in which the insulin B chain and the insulin A chain (which may contain at least one amino acid modification) are linked by a permanent linker. Single-chain insulins are described, for example, by Glidden et al. (J. Biol. Chem. 293 (2018), 47-68), or Mao et al. (Appl. Microbiol. Biotechnol. 103 (2019), 8737-8751), the contents of which are incorporated herein by reference. Single-chain insulins are also described in U.S. Patents 8,192,957; 8,501,440; 8,921,313; 8,993,516; 9,079,975; 9,200,053; 9,388,228; 9,499,600; 9,624,287; 9,758,563; 9,975,940; 10,392,429; 10,472,406; and 10,822,386, the contents of which are incorporated herein by reference. In a specific embodiment, the single-chain insulin is SCI-57, which contains a persistent hexapeptide linker GGGPRR (SEQ ID NO: 12) between the B and A chains, as described in Hua QX, Nakagawa SH, Jia W, et al., Design of an active ultrastable single-chain insulin analog: synthesis, structure, and therapeutic implications. J Biol Chem. 2008;283(21):14703-14716. doi:10.1074 / jbc.M800313200, the contents of which are incorporated herein by reference.

[0025] In one embodiment, the first domain and the second domain are directly linked to each other. In a further embodiment, the first domain and the second domain are linked to each other by a linker, for example, a linker comprising 1 to 100, particularly 10 to 60 amino acids.

[0026] The linker may be a flexible linker, such as a linker consisting of the amino acids G and S, for example a linker in which m is 1 to 5 and n is 1 to 10 (G m S) n Alternatively, the linker can be a rigid linker, e.g., comprising at least one P residue. In certain embodiments, the linker can be a cleavable linker, e.g., comprising a proteolytic cleavage site.

[0027] In certain embodiments, the fusion protein may contain additional domains, including a His tag, a purification domain such as a FLAG domain, a secretion domain, or another functional domain.

[0028] In some embodiments, the fusion protein can be conjugated to a heterologous, e.g., non-protein moiety, e.g., polyethylene glycol (PEG), or to a heterologous protein, to extend its plasma half-life. In such cases, it is advantageous to select a small conjugation partner, thereby still allowing extravasation. In a preferred embodiment, the fusion protein has a molecular weight lower than about 70 kDa, e.g., 60 kDa or less. In a further preferred embodiment, the fusion protein is not PEGylated.

[0029] The fusion proteins of the invention are useful in medicine, including veterinary and human medicine, e.g. as drugs, in the treatment of cancer, e.g. leukemia, lymphoma, hepatocellular carcinoma, melanoma, colon cancer, osteosarcoma, soft tissue sarcoma, mast cell tumor, or in the prevention or treatment of metabolic disorders, e.g. obesity or diabetes, in particular type 2 diabetes.

[0030] The fusion proteins of the invention are typically administered by injection or infusion. In a specific embodiment, administration is accompanied by co-administration of glucose, including administration of oligo- or polysaccharides, such as maltose, dextrin, starch, etc., that provide glucose to maintain sufficient glucose levels, for example, about 4.0 to about 10 mM. Additionally, administration of the fusion protein may be accompanied by some measure to compensate for the side effects of arginine depletion, such as infusion of a nitric oxide (NO) donor, such as sodium nitroprusside (SNP), and / or infusion of a hypertensive peptide, such as vasopressin, to counterbalance NO-induced vasodilation. Arginine is the only precursor for the synthesis of NO, which is short-lived. All hypertensive peptides contain arginine and are short-lived. Co-infusion of iloprost, a prostacyclin analog, has also been found to be useful in maintaining platelets.

[0031] The fusion protein may be administered as a monotherapy or in combination with an additional active agent, such as an anti-cancer agent, an anti-obesity agent, or an anti-diabetic agent. In some embodiments, the fusion protein may be co-administered with insulin, preferably with an insulin glucose clamp. In some embodiments, the fusion protein may be co-administered with an unfused amino acid enzyme that targets the same or a different amino acid as the fusion protein. In some embodiments, the arginase fusion protein may be administered with an asparaginase, such as the monomeric form of asparaginase described above, either in unfused or insulin fusion form.

[0032] The present invention improves insulin-mediated transcytosis and endocytosis of amino acid degrading enzymes by providing a fusion protein between insulin and the enzyme, instead of requiring the opportunity to capture the enzyme molecule in close proximity. Of most interest is the fusion protein between arginase and insulin, but other arginine degrading enzymes and some other enzymes that degrade other amino acids can also be fused with insulin to increase their antitumor effects.

[0033] In addition to the use of insulin and arginase fusion protein as an antitumor drug, the same fusion protein can be used to treat obesity, particularly when obesity is accompanied by diabetes already treated with insulin.By introducing arginase enzyme into fat cells, which are the main target of insulin, the growth and proliferation of fat cells is inhibited, and some of them may even die, depending on the level of intracellular arginine depletion. [Brief description of the drawings]

[0034] [Figure 1] Effect of arginase administration on plasma arginine concentration in dogs. The dashed line shows the plasma arginine concentration in two dogs without an insulin / glucose clamp. The solid line shows the plasma arginine concentration in six dogs with an insulin / glucose clamp. The combination of arginase with an insulin / glucose clamp shows a 10-fold decrease from about 100 μM to about 10 μM. [Diagram 2] Autologous partially purified liver extract rich in arginase was administered by bolus injection every 3 hours for a total of 18 hours, without (curve A) and with (curve B) an insulin / glucose clamp. Without the insulin / glucose clamp, plasma arginine levels fell to near zero and returned to normal levels before the next bolus injection 3 hours later. With the insulin / glucose clamp, plasma arginine fell to below detection and remained there for 18 hours. [Diagram 3] A fusion protein having human liver arginase (ARG-1) as a first domain containing a histidine tag and human proinsulin as a second domain linked by a flexible linker. [Figure 4] A fusion protein having human liver arginase (ARG-1) as the first domain containing a histidine tag after disulfide bridges and cleavage of the proinsulin C-peptide, and human insulin as the second domain linked by a flexible linker. [Diagram 5] A fusion protein with human liver arginase (ARG-1) as the first domain containing a histidine tag and human proinsulin as the second domain connected by a flexible linker, showing the C-peptide cleavage sites by PC1 / 3 and cpE enzymes. [Figure 6] A fusion protein having human liver arginase (ARG-1) as the first domain containing a histidine tag, and a single-chain insulin analog (SCI-57) with a permanent hexapeptide linker (C-linker) between the B and A chains containing four favorable amino acid substitutions (at B10, B28, B29, and A8). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] [Experimental observations and conclusions therefrom] Our studies of systemic depletion of arginine and asparagine as anticancer therapy, conducted in healthy experimental dogs and several dogs with cancer since 1995 and ongoing, have provided strong evidence for a role for insulin in increasing the efficacy of these therapies that depend on the enzymatic degradation of targeted amino acids.

[0036] In the first phase of this project, extracorporeal removal of targeted amino acids was performed by selective dialysis. Using a modified dialysis apparatus, blood was dialyzed against a dialysate containing most of the known low molecular weight water-soluble components of plasma (a total of more than 52 electrolytes) except for the targeted amino acids. The effectiveness of the process was verified by measuring all amino acids at the inlet and outlet of the dialysis filter. Most essential amino acids were targeted one by one in these experiments, among which arginine was of main interest due to its established depletion effect on various tumor lines tested in vitro. However, continuous dialysis over several days failed to significantly reduce the plasma concentration of any essential amino acid, even though the targeted amino acids were almost completely washed out by the filter. Target amino acid concentrations at the filter outlet were below detection, while concentrations at the inlet remained close to normal values. Blood flow rates were very high, up to 300 ml / min in a dog weighing about 30 kg. The failure of this approach was precisely due to the homeostatic control of essential amino acids, which resulted in an estimated loss of as much as 10% of the total body protein per day.

[0037] In subsequent experimental studies, the inventors decided to use an insulin / glucose clamp to inhibit protein degradation and promote protein synthesis. While using selective dialysis with the same parameters, the plasma arginine concentration could now be reduced by about 10-fold from about 100 μM to about 10 μM, as shown in FIG. 1. The dashed line shows the plasma arginine concentration in two dogs without an insulin / glucose clamp. The solid line shows the plasma arginine in six dogs with an insulin / glucose clamp.

[0038] However, sampling of the lymphatic system did not show a decrease in arginine concentration, which was about 200 μM, even higher than the normal plasma concentration. The conclusion was clear - while dialysis can reduce the arginine concentration in plasma, molecular exchange by diffusion and convective transport between blood and interstitial fluid cannot compensate for the influx of amino acids from the endogenous protein metabolism, mainly from muscle proteins.

[0039] The inventors then decided to use enzymatic degradation of targeted amino acids. In the first approach targeted to the removal of arginine, the inventors used a partially purified liver extract rich in arginase. In preclinical experiments in healthy dogs, autologous liver extract was delivered by bolus injection every 3 hours for a total of 18 hours. Without insulin / glucose clamp, plasma arginine levels fell to near zero and returned to normal levels before the next bolus injection 3 hours later (Figure 2, curve A). With insulin / glucose clamp, plasma arginine fell to below detection and remained there for the entire 18-hour experimental period, even though the extract was still delivered by bolus injection (Figure 2, curve B).

[0040] After these pilot experiments, the inventors decided to use continuous infusions of enzymatically active material from various sources, including recombinant enzymes.

[0041] The rapid fall and increase in plasma arginine by bolus infusion without an insulin / glucose clamp was forgotten until several years later, when there is some further evidence of the insulin effect in this anticancer treatment modality.

[0042] In over 100 sessions in healthy experimental dogs and a small number of dogs with cancer, the inventors noted loss of albumin and moderate edema accompanying the development of the insulin / glucose clamp. The loss of plasma albumin was due to protein turnover and the edema was due to an undesirable but not limiting side effect.

[0043] However, closer examination of previously observed arginine oscillations induced by bolus injection of liver extracts provided clues to a new and unexpected role for insulin in addition to its effects in regulating protein turnover.

[0044] Hepatic arginase is a monomer with a molecular weight of about 35 kDa, exactly in the middle of the molecular weight range that glomerular filtration can remove from plasma (up to about 70 kDa). Albumin has a molecular weight of 72 kDa, and its loss by diffusion into the extravascular fluid or by glomerular filtration is negligible. However, insulin is known to increase capillary permeability, and thus maintaining insulin concentrations above physiological concentrations for long periods of time allows some extravasation of albumin, which causes edema. As mentioned before, this was not limiting to the protocol and could be compensated for by using standard diuretics.

[0045] However, the role of insulin was crucial to make arginase-induced arginine depletion an effective mechanism that does not remain in the vasculature. Arginase, being 35 kDa, is rapidly cleared by glomerular filtration. The use of an insulin / glucose clamp causes an increase in capillary permeability sufficient to cause arginase extravasation, thus protecting it from removal by the kidney. It also delivers the enzyme to the interstitial fluid, where the true effect of arginine depletion on cancer must lie. Removal of arginine from plasma is an insufficient substitute for an enzymatic anticancer effect. In all current clinical trials as anticancer treatments, arginase or arginine deiminase are PEGylated. PEGylation of these enzymes prevents their extravasation, which explains the lack of clinical success despite removal of arginine from plasma.

[0046] These observations provide a plausible explanation for the role of insulin / glucose in arginase extravasation.

[0047] Our in vitro study using canine cancer cell lines (Wells JW, Evans CH, Scott MC, Rutgen BC, O'Brien TD, Modiano JF, Cvetkovic G, Tepic S. Arginase treatment prevents the recovery of canine lymphoma and osteosarcoma cells resistant to the toxic effects of prolonged arginine deprivation. PLoS One. 2013;8(1):e54464. doi: 10.1371 / journal.pone.0054464. Epub 2013 Jan 24. PMID: 23365669; PMCID: PMC3554772.) suggested that the remarkable effect of arginase in rapidly killing cancer cells is due in part to its attachment to or entry into cancer cells. Depletion of arginine in the extracellular environment was necessary but not sufficient. Selectivity for cancer cells over healthy cells may be due in part to the increased rate of endocytosis exhibited by cancer cells.

[0048] Cancer and rapidly proliferating healthy cells are known to have high expression of insulin receptors. However, in contrast to cancer cells, healthy cells respond to arginine depletion by arresting their cell cycle and can survive for up to three weeks in a quiescent state. In contrast, the lack of cycle control, which is a hallmark of all cancers, most often leads them to metabolic death.

[0049] The present invention aims to take advantage of previous findings by providing a fusion protein of insulin and arginase, specifically human insulin and human liver arginase (arginase-1). By using an appropriate linker in the fusion, the activity of both insulin and arginase is maintained.

[0050] In the in vivo studies we have performed so far, the average effective doses of insulin and arginase were close to stochiometric. However, if further in vivo studies with fusion proteins show differences in the efficiency of fusion proteins versus single proteins, a combination of the fusion molecule with regular insulin and / or regular arginase could be used. EXAMPLES

[0051] [Example 1] A fusion protein is provided that includes human liver arginase (UniProtKB, arginase-1, P05089) as the N-terminal domain and human proinsulin as the C-terminal domain. Human proinsulin is fused to arginase at its N-terminus, the start of the B chain (UniProtKB, human insulin, P01308).

[0052] For this purpose, suitable host cells, e.g., prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, or mammalian cells, are transfected with a nucleic acid molecule encoding the fusion protein operably linked to expression control sequences compatible with the respective host cell. The host cells are cultured under conditions suitable for expression of the fusion protein. The fusion protein is purified from the cells or from the culture medium according to known techniques.

[0053] After recombinant production, the B and A chains are cross-linked via a disulfide bond and the C-peptide is enzymatically removed.

[0054] The following sequence of ARG-1 is histidine tagged for affinity purification, has a full length of 331 amino acids, and was used by the inventors to produce highly active human liver arginase in E. coli:

[0055] MGHHHHHGSSAKSRTIGIIGAPFSKGQPRGGVEEGPTVTLRKAGLLEKLKEQECDVKDYGDLPFADIPNDSPFQIVKNPRSVGKASEQLAGKVAEVKKNGRISLVLGGDHSLAIGSISGHARVHPDLGVIWVDAHTDINTPLTTTSGNLHGQPVSFLLKELKGKIPDVPGFSWVTPCISAKDIVYIGLRDVDPGEHYILKTLGIKYFSMTEVDRLGIGKVMEETLSYLLGRKKRPIHLSFDVDGLDPSFTPATGTPVVGGLTYREGLYITEEIYKTGLLSGLDIMEVNPSLGKTPEEVTRVNTAVAITLACFGLAREGNHKPIDYLNPPK (SEQ ID NO: 1)

[0056] The proinsulin sequence of human insulin: FVNQHLCGSHLVEALYLVCGERGFFYTPKT RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR GIVEQCCTSICSLYQLENYCN (SEQ ID NO:2) is 86 amino acids long.

[0057] The B-chain sequence at the start of the proinsulin sequence: FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 3) is 30 amino acids long.

[0058] C-peptide: RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR (SEQ ID NO: 4) (underlined in the proinsulin sequence above) is 35 amino acids long and is enzymatically removed after cross-linking of the B and A chains.

[0059] A strand sequence: GIVEQCCTSICSLYQLENYCN (SEQ ID NO:5) is 21 amino acids long.

[0060] Disulfide bonds link the cysteine ​​residues at position 7 of both the B and A chains, and between cysteine ​​residues at positions 19 of the B chain and 20 of the A chain, with an additional disulfide bond between cysteine ​​residues at positions 6 and 11 in the A chain.

[0061] The first three (FVN) and the last four (TPKT) amino acids of the B chain are known not to interact with either the A chain or the insulin receptor, in contrast only the last amino acid (N) of the A chain is known not to participate in any interaction.

[0062] A linker X can be inserted between the C-terminus of arginase (K) and the N-terminus of the B chain of proinsulin (F). A flexible linker of the GS type is the first choice since only the three amino acids at the N-terminus of the B chain are known not to participate in any interactions. A linker Y, e.g. a short GS linker, can be incorporated at the N-terminus (S) of arginase to link a purification tag, e.g. a His tag, to the arginase.

[0063] One of the most commonly used GS-type linkers is (G4S). n The length of the linker follows its expected function, in this case to improve the spatial separation of arginase and insulin and maintain their independent activities. In one embodiment, (G4S) 10 (SEQ ID NO: 6) is used.

[0064] In a specific embodiment, the amino acid sequence of the fusion protein comprising a His purification tag, human Arginase I, and proinsulin is as follows (Figure 3):

[0065] MGHHHHHH*Y*SAKSRTIGIIGAPFSKGQPRGGVEEGPTVLRKAGLLEKLKEQECDVKDYGDLPFADIPNDSPFQIVKNPRSVGKASEQLAGKVAEVKKNGRISLVLGGDHSLAIGSISGHARVHPDLGVIWVDAHTDINTPLTTTSGNLHGQPVSFLLKELKGKIPDVPGFSWVTPCISAK DIVYIGLRDVDPGEHYILKTLGIKYFSMTEVDRLGIGKVMEETLSYLLGRKKRPIHLSFDVDGLDPSFTPATGTPVVGGLTYREGLYITEEIYKTGLLSGLDIMEVNPSLGKTPEEVTRTVNTAVAITLACFGLAREGNHKPIDYLNPPK*X*FVNQHLCGSHLVEALYLVCGERGFFYTPKT RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 7), where Y is a linker, specifically a GS linker, and X is a linker, specifically (GGGGS) n is a linker, and n is 1 to 10.

[0066] The fusion protein after removal of the C-peptide consists of two polypeptides, polypeptide (i) consisting of the His tag, the arginase domain, a linker, and the insulin B peptide, and polypeptide (ii) consisting of the insulin A chain, and polypeptides (i) and (ii) are cross-linked by a disulfide bond, see below (Figure 4):

[0067] Polypeptide (i): MGHHHHHH*Y*SAKSRTIGIIGAPFSKGQPRGGVEEGPTVLRKAGLLEKLKEQECDVKDYGDLPFADIPNDSPFQIVKNPRSVGKASEQLAGKVAEVKKNGRISLVLGGDHSLAIGSISGHARVHPDLGVIWVDAHTDINTPLTTTSGNLHGQPVSFLLKELKGKIPDVPGFSWVTPCISAKDIVY IGLRDVDPGEHYILKTLGIKYFSMTEVDRLGIGKVMEETLSYLLGRKKRPIHLSFDVDGLDPSFTPATGTPVVGGLTYREGLYITEEIYKTGLLSGLDIMEVNPSLGKTPEEVTRTVNTAVAITLACFGLAREGNHKPIDYLNPPK*X*FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 8), where Y is a linker, specifically a GS linker, and X is a linker, specifically (GGGGS) n is a linker, and n is 1 to 10.

[0068] Polypeptide (ii): GIVEQCCTSICSLYQLENYCN (SEQ ID NO:5)

[0069] The enzymatic removal of the disulfide bridges of the insulin chains and the C-peptide is preferably carried out with the fusion protein bound to a purification material, such as a purification column or filter, by means of its affinity tag. In one embodiment, the purification material is an affinity filter or column, such as a Ni filter or column, to which the fusion protein is bound by its His tag (Figure 5).

[0070] The length of the fusion protein with the G4S linker (SEQ ID NO: 9) is 331+5+30+21=387 residues, with a molecular weight of approximately 42.0 kDa.

[0071] (G4S) 10 (SEQ ID NO:6) With the linker it is 432 residues in length and has a molecular weight of approximately 45.6 kDa.

[0072] Assuming that the increase in vasculature permeability by insulin is the result of its transport through endothelial cells, possibly by transcytosis that also leads to the extravasation of the 35.8 kDa arginase (with a His tag), it is reasonable to expect that the rate of transcytosis of the approximately 42-46 kDa fusion protein would be similar, but potentially even higher, due to the "tag-along" effect of insulin on arginase.

[0073] The same is true for entry into target cells via endocytosis, specifically into cancer cells that are known to overexpress the insulin receptor: in the many in vivo experiments we performed using arginase and insulin, we found no significant adverse effects on healthy cells—only a transient decrease in their proliferation rate.

[0074] [Example 2] A further embodiment of the invention is a fusion protein of insulin with human arginase-2 (also known as arginase II, kidney-type arginase, non-hepatic arginase), which is found primarily in the kidney and some other tissues but not in the liver. Arginase-2 (UniProt P78540) is 354 residues long and has a molecular weight of 38578 Da. If the same His tag (GHHHHHHGS) (SEQ ID NO: 10) as Arginase-1 is used at the N-terminus, the protein will have 363 residues and a molecular weight of approximately 39.7 kDa. (G4S) as described above n When a linker is used, the fusion protein of human arginase-2 and insulin has a molecular weight of 46 to 50 kDa.

[0075] [Example 3] A further embodiment of the invention is a fusion protein of insulin and human cobalt-substituted arginase I or arginase II, which may be produced by fermenting E. coli cells expressing the fusion protein to produce the fusion protein, substituting cobalt for manganese in the recombinant protein to provide a Co-substituted fusion protein, which is further purified, as described in US 20121 / 0189371 supra.

[0076] [Example 4] A further embodiment of the invention is a fusion protein of human Arginase-1 and SCI-57 (Figure 6). In addition to the persistent hexapeptide C-linker GGGPRR (SEQ ID NO: 12) that replaces the C-peptide of natural proinsulin, the B-chain (SEQ ID NO: 11) and A-chain (SEQ ID NO: 13) of SCI-57 contain further modifications to human insulin, namely four substitutions: Thr A8 to His, His B10 to Asp, Pro B28 to Asp, and Lys B29 to Pro and is shown in grey in Figure 6. SCI-57 (SEQ ID NO: 14) is an ultrastable single-chain insulin with high binding affinity to the insulin receptor. Of particular interest in the present invention is the use of a persistent peptide linker in SCI-57, allowing the production of a fusion protein with monomeric arginase in a single step.

[0077] [Example 5] The fusion protein of the present invention is administered to patients by injection.For the systemic and deep depletion required in antitumor therapy, the delivery of the fusion protein is preferably performed together with the simultaneous injection of glucose, and may be performed together with additional measures such as the simultaneous injection of nitric oxide donor (e.g., SNP) and / or vasopressin (e.g., arginine-vasopressin) to compensate for the side effects of low arginine.

[0078] Lower doses may be necessary to treat obesity and / or type 2 diabetes, with doses closer to the standard use of insulin alone.

[0079] The sequences listed as SEQ ID NO:1 to SEQ ID NO:14 according to the present invention are defined as follows:

[0080] SEQ ID NO:1 (ARG-1 with histidine tag) Met Gly His His His His His His Gly Ser Ser Ala Lys Ser Arg Thr Ile Gly Ile Ile Gly Ala Pro Phe Ser Lys Gly Gln Pro Arg Gly Gly Val Glu Glu Gly Pro Thr Val Leu Arg Lys Ala Gly Leu Leu Glu Lys Leu Lys Glu Gln Glu Cys Asp Val Lys Asp Tyr Gly Asp Leu Pro Phe Ala Asp Ile Pro Asn Asp Ser Pro Phe Gln Ile Val Lys Asn Pro Arg Ser Val Gly Lys Ala Ser Glu Gln Leu Ala Gly Lys Val Ala Glu Val Lys Lys Asn Gly Arg Ile Ser Leu Val Leu Gly Gly Asp His Ser Leu Ala Ile Gly Ser Ile Ser Gly His Ala Arg Val His Pro Asp Leu Gly Val Ile Trp Val Asp Ala His Thr Asp Ile Asn Thr Pro Leu Thr Thr Thr Ser Gly Asn Leu His Gly Gln Pro Val Ser Phe Leu Leu Lys Glu Leu Lys Gly Lys Ile Pro Asp Val Pro Gly Phe Ser Trp Val Thr Pro Cys Ile Ser Ala Lys Asp Ile Val Tyr Ile Gly Leu Arg Asp Val Asp Pro Gly Glu His Tyr Ile Leu Lys Thr Leu Gly Ile Lys Tyr Phe Ser Met Thr Glu Val Asp Arg Leu Gly Ile Gly Lys Val Met Glu Glu Thr Leu Ser Tyr Leu Leu Gly Arg Lys Lys Arg Pro Ile His Leu Ser Phe Asp Val Asp Gly Leu Asp Pro Ser Phe Thr Pro Ala Thr Gly Thr Pro Val Val Gly Gly Leu Thr Tyr Arg Glu Gly Leu Tyr Ile Thr Glu Glu Ile Tyr Lys Thr Gly Leu Leu Ser Gly Leu Asp Ile Met Glu Val Asn Pro Ser Leu Gly Lys Thr Pro Glu Glu Val Thr Arg Thr Val Asn Thr Ala Val Ala Ile Thr Leu Ala Cys Phe Gly Leu Ala Arg Glu Gly Asn His Lys Pro Ile Asp Tyr Leu Asn Pro Pro Lys

[0081] SEQ ID NO:2 (human protein) Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn

[0082] SEQ ID NO:3 (B chain) Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr

[0083] SEQ ID NO:4 (C-peptide) Arg Arg Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg

[0084] SEQ ID NO:5 (A chain / polypeptide (ii)) Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn

[0085] SEQ ID NO:6 ((G4S)10 linker) Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

[0086] SEQ ID NO:7 (artificial sequence) Xaa at position 9 = linker, specifically GS linker Xaa at position 331 = linker, specifically (GGGGS)n linker, n is 1 to 10 Met Gly His His His His His Xaa Ser Ala Lys Ser Arg Thr Ile Gly Ile Ile Gly Ala Pro Phe Ser Lys Gly Gln Pro Arg Gly Gly Val Glu Glu Gly Pro Thr Val Leu Arg Lys Ala Gly Leu Leu Glu Lys Leu Lys Glu Gln Glu Cys Asp Val Lys Asp Tyr Gly Asp Leu Pro Phe Ala Asp Ile Pro Asn Asp Ser Pro Phe Gln Ile Val Lys Asn Pro Arg Ser Val Gly Lys Ala Ser Glu Gln Leu Ala Gly Lys Val Ala Glu Val Lys Lys Asn Gly Arg Ile Ser Leu Val Leu Gly Gly Asp His Ser Leu Ala Ile Gly Ser Ile Ser Gly His Ala Arg Val His Pro Asp Leu Gly Val Ile Trp Val Asp Ala His Thr Asp Ile Asn Thr Pro Leu Thr Thr Thr Ser Gly Asn Leu His Gly Gln Pro Val Ser Phe Leu Leu Lys Glu Leu Lys Gly Lys Ile Pro Asp Val Pro Gly Phe Ser Trp Val Thr Pro Cys Ile Ser Ala Lys Asp Ile Val Tyr Ile Gly Leu Arg Asp Val Asp Pro Gly Glu His Tyr Ile Leu Lys Thr Leu Gly Ile Lys Tyr Phe Ser Met Thr Glu Val Asp Arg Leu Gly Ile Gly Lys Val Met Glu Glu Thr Leu Ser Tyr Leu Leu Gly Arg Lys Lys Arg Pro Ile His Leu Ser Phe Asp Val Asp Gly Leu Asp Pro Ser Phe Thr Pro Ala Thr Gly Thr Pro Val Val Gly Gly Leu Thr Tyr Arg Glu Gly Leu Tyr Ile Thr Glu Glu Ile Tyr Lys Thr Gly Leu Leu Ser Gly Leu Asp Ile Met Glu Val Asn Pro Ser Leu Gly Lys Thr Pro Glu Glu Val Thr Arg Thr Val Asn Thr Ala Val Ala Ile Thr Leu Ala Cys Phe Gly Leu Ala Arg Glu Gly Asn His Lys Pro Ile Asp Tyr Leu Asn Pro Pro Lys Xaa Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn

[0087] SEQ ID NO:8 (Polypeptide (i)) Xaa at position 9 = linker, specifically a GS linker Xaa at position 331 = linker, specifically a (GGGGS)n linker, where n ranges from 1 to 10 Met Gly His His His His His His Xaa Ser Ala Lys Ser Arg Thr Ile Gly Ile Ile Gly Ala Pro Phe Ser Lys Gly Gln Pro Arg Gly Gly Val Glu Glu Gly Pro Thr Val Leu Arg Lys Ala Gly Leu Leu Glu Lys Leu Lys Glu Gln Glu Cys Asp Val Lys Asp Tyr Gly Asp Leu Pro Phe Ala Asp Ile Pro Asn Asp Ser Pro Phe Gln Ile Val Lys Asn Pro Arg Ser Val Gly Lys Ala Ser Glu Gln Leu Ala Gly Lys Val Ala Glu Val Lys Lys Asn Gly Arg Ile Ser Leu Val Leu Gly Gly Asp His Ser Leu Ala Ile Gly Ser Ile Ser Gly His Ala Arg Val His Pro Asp Leu Gly Val Ile Trp Val Asp Ala His Thr Asp Ile Asn Thr Pro Leu Thr Thr Thr Ser Gly Asn Leu His Gly Gln Pro Val Ser Phe Leu Leu Lys Glu Leu Lys Gly Lys Ile Pro Asp Val Pro Gly Phe Ser Trp Val Thr Pro Cys Ile Ser Ala Lys Asp Ile Val Tyr Ile Gly Leu Arg Asp Val Asp Pro Gly Glu His Tyr Ile Leu Lys Thr Leu Gly Ile Lys Tyr Phe Ser Met Thr Glu Val Asp Arg Leu Gly Ile Gly Lys Val Met Glu Glu Thr Leu Ser Tyr Leu Leu Gly Arg Lys Lys Arg Pro Ile His Leu Ser Phe Asp Val Asp Gly Leu Asp Pro Ser Phe Thr Pro Ala Thr Gly Thr Pro Val Val Gly Gly Leu Thr Tyr Arg Glu Gly Leu Tyr Ile Thr Glu Glu Ile Tyr Lys Thr Gly Leu Leu Ser Gly Leu Asp Ile Met Glu Val Asn Pro Ser Leu Gly Lys Thr Pro Glu Glu Val Thr Arg Thr Val Asn Thr Ala Val Ala Ile Thr Leu Ala Cys Phe Gly Leu Ala Arg Glu Gly Asn His Lys Pro Ile Asp Tyr Leu Asn Pro Pro Lys Xaa Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr

[0088] SEQ ID NO:9 (G4S linker) Gly Gly Gly Gly Ser

[0089] SEQ ID NO: 10 (His tag) Gly His His His His His His Gly Ser

[0090] SEQ ID NO: 11 (B chain) Phe Val Asn Gln His Leu Cys Gly Ser Asp Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Asp Pro Thr

[0091] SEQ ID NO:12 (C-linker) Gly Gly Gly Pro Arg Arg

[0092] SEQ ID NO: 13 (A chain) Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn

[0093] SEQ ID NO:14 (SCI-57) Phe Val Asn Gln His Leu Cys Gly Ser Asp Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Asp Pro Thr Gly Gly Gly Pro Arg Arg Gly Ile Val Glu Gln Cys Cys His Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn

Claims

1. A fusion protein comprising a first domain and a second domain linked to each other by a linker, the first domain comprises human arginase and the second domain comprises insulin; the first domain (enzyme) is located N-terminal to the second domain (insulin); The linker may be a flexible linker, e.g., a linker consisting of amino acids G and S, e.g., a (G m S) n linker where m is 1-5 and n is 1-10, a rigid linker, or a cleavable linker; Fusion proteins.

2. 2. The fusion protein of claim 1 , The fusion protein is a gene fusion. Fusion proteins.

3. 3. The fusion protein according to claim 1 or 2, The human arginase is human liver arginase (human arginase-1) or human kidney arginase (human arginase-2). Fusion proteins.

4. The fusion protein according to any one of claims 1 to 3, The human arginase is a monomeric protein, e.g., a monomeric arginase. Fusion proteins.

5. The fusion protein according to any one of claims 1 to 4, The insulin is human insulin or an insulin analogue, including a single-chain insulin. Fusion proteins.

6. Encoding the fusion protein according to any one of claims 1 to 5, Nucleic acid molecule.

7. Transfected with the nucleic acid molecule of claim 6, host cell.

8. A method for producing a fusion protein according to any one of claims 1 to 5, comprising:

10. A method of producing a fusion protein comprising culturing a host cell according to claim 7 and obtaining the fusion protein from the host cell or from the culture medium. method.

9. A fusion protein according to any one of claims 1 to 5, For medical use Fusion proteins.

10. A fusion protein according to any one of claims 1 to 5, For use as a medicament in the treatment of cancer or in the prevention or treatment of metabolic disorders such as obesity or diabetes, particularly type 2 diabetes, Fusion proteins.

11. A fusion protein according to any one of claims 1 to 5 for the use according to claim 9 or 10, the administration of the fusion protein is accompanied by the simultaneous administration of glucose; and Optionally, measures to compensate for the side effects of arginine depletion may be taken. Fusion proteins.

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