JP2013253079A - BIOLOGICALLY ACTIVE Fc FUSION PROTEIN OF HUMAN URINARY TRYPSIN INHIBITOR, AND PREPARATION METHOD AND APPLICATION THEREOF - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a recombinant Fc fusion protein of a human urinary trypsin inhibitor, the protein having biological activity similar with that of the human urinary trypsin inhibitor (hUTI).SOLUTION: There is provided: a recombinant hUTI-L-Fc fusion protein, comprising hUTI, about 20 or less flexible peptide linkers, and human IgG Fc or its variant (vFc); and a preparation method of the fusion protein with high expression level. The Fc variant of IgG is preferably insoluble and shows side effects mediated by minimum unwanted Fc. The recombinant hUTI-L-Fc fusion protein has sufficient biological activity and/or an extended or elongated half-life period in serum. Thus improved pharmacokinetics and/or pharmacodynamics are/is provided, thereby using different or smaller dosage amount and/or achieving better or different therapeutic efficacy.

Description

Background Human urinary trypsin inhibitor (human UTI or hUTI), also called urinastatin or bikunin, is a glycosylated protein that contains two Kunitz-type protease inhibitor domains. Human UTI is found in human urine and blood and has a molecular weight of about 40 kilodaltons (kD). Human UTI is known to have many physiological actions, such as an inhibitory effect on the activity of serine proteases including trypsin, α-chymotrypsin, plasmin, cathepsin G and leukocyte elastase. Human UTI also immunizes by down-regulating the release of pro-inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin-1 (IL-1) and interleukin-6 (IL-6). Show regulatory effects (eg Park, et al. J. Korean Med. Sci., 25: 128-34, 2010; Sato et al. Jpn. J. Thorac. Cardiovasc. Surg., 48: 428-34, 2000 Park et al. Korean J. Anesthesiol., 58: 334-337, 2010). Furthermore, hUTI inhibits malignant mesothelioma cell migration by neutralizing PDGF-D active dimer (PDGF-DD) / PDGF-bbR mediated signaling and Significantly reduced mammary tumors in breast cancer xenograft nude mouse models by inhibiting proliferation and down-regulating CXCL4 and MMP-9 expression (eg, Yaguchi et al. Cancer Lett 288: 214-218, 2010; Sun et al. J. Int. Med. Res. 38: 967-976, 2010), suggesting a possible role of hUTI in cancer therapy.

  Furthermore, hUTI has been shown to act to suppress the proteolytic action of trypsin on various tissues and to exert local anti-inflammatory effects. When used in cases of general anesthesia in high-risk patients, intravenous infusion of hUTI can improve perfusion to many organs and reduce the inflammatory response. Therefore, hUTI is indicated for acute inflammatory diseases including acute pancreatitis, systemic inflammatory response syndrome, circulatory dysfunction, disseminated intravascular coagulation (DIC) and multiple organ failure (eg, Jong In Han , Korean J. Anesthesiol., 58: 325-327, 2010).

  In animal model studies, hUTI has been reported to have protective effects against endotoxin-induced shock as well as hepatic ischemia-reperfusion injury (eg, Inoue et al. Expert Opin. Investig. Drugs, 19: 513-20, 2010; Wu et al. Hepatobiliary Pancreat. Dis. Int., 8: 53-58, 2009). In clinical studies, the combined use of hUTI and thymosin alpha-1 increased the survival of patients with severe sepsis (see, eg, Li et al. J. Intensive Care Med., 24: 47-53, 2009) . In another clinical study, hUTI has been shown to be effective in treating acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) induced by SARS virus (eg, US Patent No. 7,470,666).

  The trade name “miraclide” for hUTI purified from human urine was first marketed in 1985 by Mochida Pharmaceutical, Japan. “Miracrid” has demonstrated clinical efficacy as a drug for the treatment of acute pancreatitis and acute circulatory failure caused by shock. Other similar products are Techpool Biopharma, China (“Urinastatin for Injection”), Han Lim Pharmaceutical, Korea (Ulistin), Kolon Pharmaceutical, Korea (Ustatin) and Yu Young Pharmaceutical, Korea (statin ( Statin)). In Japan and China, hUTI is widely used as a drug for the treatment of acute pancreatitis, disseminated intravascular coagulation (DIC) and shocks such as hemorrhagic shock, septic shock, traumatic shock and burn shock.

  HUTI pharmaceutical grade products on the market are purified from human urine. This supply of starting material can be unpredictable and possible contamination by human pathogens is dangerous. Through the use of recombinant DNA technology, rhUTI was produced in yeast and the purified material was shown to have trypsin inhibitory activity in vitro. Recombinant hUTI protein expression in yeast has been reported to be as low as 55 mg / L (see, eg, Jian-qiu Wang et al, Protein Expr. Purif., 60: 127-31, 2008; US Patent No. 5,407,915). In addition, recombinant proteins produced in yeast cells tend to undergo different post-translational modifications, thereby undesired side effects such as triggering potentially reduced potency and / or immune responses to therapeutic proteins in vivo. Is brought about. Therefore, rhUTI protein derived from yeast may not be a good candidate for biological therapeutics (Brooks SA, Mol. Biotechnol., 28: 241-55, 2004).

  The ability to make complex post-translational modifications is a major reason that most biotherapeutic agents are produced in mammalian cell cultures. In fact, only a few biopharmaceutical proteins such as albumin (Recombumin®, manufactured by Novozymes Biopharma) and fast-acting insulin analogues (Humalog®, manufactured by Eli Lilly) are subject to simple modifications Can be produced using yeast. In general, recombinant proteins produced in mammalian cells are fully folded and appropriately glycosylated. However, rhUTI expression in Chinese hamster ovary (CHO) cells has been reported to produce extensive protein aggregates in cell culture, resulting in reduced activity. Protein aggregates have been shown to be covalently crosslinked through non-natural disulfide bonds (see, eg, Seefeldt MB et al. Protein Sci., 13: 2639-50, 2004).

  In healthy volunteers, the plasma half-life of intravenous (iv) hUTI between 0 and 3 hours after injection is about 33 minutes, indicating about 90% loss of injected hUTI. Occupy. During the subsequent 4 hours, a slower second phase was observed with a half-life of about 2 hours. Seven hours after injection, only 1% of free hUTI remained in the plasma (see, eg, Jonsson-Berling et al. Scand. J. Clin. Lab. Invest., 51: 549-57, 1991). Organ tissue distribution studies in rats have shown that the kidney is the primary site for hUTI degradation and excretion. Furthermore, the distribution and localization of hUTI in normal and malignant human tissues using immunohistochemical techniques found hUTI in the proximal tubule of the kidney (see, eg, Yoshida et al. Cancer, 64: 860- 9, 1989).

  Although the detailed mechanism involved in hUTI clearance remains unclear, the kidney appears to be a major site of hUTI catabolism. In order to prolong the half-life of hUTI in the circulation, the excretion through the kidney can be slowed by increasing the molecular weight of hUTI. Such an approach has been taken in the design of long acting drugs, for example adding extra N-glycosylation sites to EPO (Aranesp®, manufactured by Amgen) or adding PEG molecules to G-CSF. Addition (Neulasta®, manufactured by Amgen) and the like. By adding human IgG Fc or a variant thereof to the C-terminus of hUTI, the resulting UTI-Fc molecule will not only have a higher molecular weight, but will also exhibit a longer half-life for human IgG Fc in plasma .

  The IgG class of immunoglobulins is one of the most abundant proteins in human blood. Its circulating half-life can reach as long as 21 days. Fusion proteins have been reported to combine the Fc region of IgG with other protein domains (eg, various cytokines and soluble receptors) (eg, Capon et al., Nature, 337: 525-531, 1989; Chamow et al., Trends Biotechnol., 14: 52-60, 1996); US Pat. Nos. 5,116,964 and 5,541,087). The prototype of the fusion protein is a homodimeric protein linked through a cysteine residue in the hinge region of IgG Fc, resulting in a molecule similar to an IgG molecule that does not contain a CH1 domain and light chain. Due to structural homology, Fc fusion proteins exhibit a pharmacokinetic profile equivalent to human IgG with similar isotypes in vivo. This approach has been applied to several therapeutically important cytokines such as EPO, G-CSF, and soluble receptors such as TNF-Rc and IL-5-Rc (see, eg, US Patent Nos., 5,349,053 and 7,232,668). Therefore, prolonging or altering the hUTI circulating half-life by producing a fusion protein comprising hUTI linked to the Fc portion of a human IgG (IgG1, IgG2, IgG4) protein as disclosed or described in the present invention It may be desirable to increase / modify and / or its biological activity. It is also desirable and part of the present invention to produce such an alternative to hUTI that exhibits different and / or more desirable clinical and / or pharmaceutical properties.

  Furthermore, in most reported Fc fusion protein molecules, the hinge region acts as a spacer between the Fc region and the amino-terminal cytokine or soluble receptor, so that these two parts of the molecule function separately (See, for example, Ashkenazi et al., Current Opinion in Immunology, 9: 195-200, 1997). It is therefore desirable to have a suitable linker that acts as such a spacer and is part of the present invention so that both UTI domains can interact independently with a serine protease.

  The Fc region of human immunoglobulin plays an important role in immune defense for the elimination of pathogens. The effector functions of IgG are mediated by the Fc region through two major mechanisms: (1) phagocytosis of pathogen uptake or antibody-dependent cellular cytotoxicity (ADCC) by binding to cell surface Fc receptors (FcγRs) Lysis by killer cells can occur via the pathway, or (2) binding of the first complement component C1 to the C1q moiety initiates the complement dependent cytotoxicity (CDC) pathway and pathogen lysis occurs . Of the four human IgG isotypes, IgG1 and IgG3 are effective in binding to FcγR. The binding affinity of IgG4 to FcγR is about an order of magnitude lower than IgG1 or IgG3, while IgG2 binding to FcγR is less than detection. Human IgG1 and IgG3 are also effective in binding to C1q and activating the complement cascade. Human IgG2 binds only weakly to complement, and IgG4 appears to be very poorly capable of activating the complement cascade (eg Jefferis et al., Immunol. Rev., 163: 59- 76, 1998). In therapeutic use, if hUTI-L-Fc binds to various proteases, whether the Fc region of the fusion protein mediates or does not mediate an effector function that causes lysis or removal of cells with protease bound to the cell surface Will not be definitive or important. Thus, it is clear from the invention disclosed herein that the FUT region of hUTI-Fc can be a native IgG Fc or an insoluble Fc variant. As discussed above, native IgG Fc mediates various levels of effector function. In contrast, non-soluble Fc is inactive with respect to binding to FcγRs and C1q to trigger effector function. In order to obtain non-soluble Fc, certain amino acids in the native Fc region must be mutated to attenuate effector function.

  By comparing the amino acid sequences of the human and mouse IgG isotypes, the Fc portion near the N-terminus of the CH2 domain is involved in playing a role in the binding of IgG Fc to FcγRs. The importance of the motifs at positions 234 to 237 has been demonstrated using genetically engineered antibodies (see, eg, Duncan et al., Nature, 332: 563-564, 1988). The numbering of amino acid residues follows the EU index described in Kabat et al. (Sequences of Proteins of Immunological Interest, 5th Edition, United States Department of Health and Human Services, 1991). Of the four human IgG isotypes, IgG1 and IgG3 bind best to FcγRs and share the sequence of Leu234-Leu-Gly-Gly237 (IgG3 is not shown in FIG. 1). In IgG4, which binds FcγRs with lower affinity, this sequence contains a single amino acid substitution, namely Phe for Leu at position 234. In IgG2, which does not bind to FcγRs, there are two substitutions and one deletion, resulting in Val234-Ala-Gly237 (FIG. 1). In order to minimize Fc binding to FcγR and thus minimize ADCC activity, Leu235 in IgG4 was replaced by Ala (eg, Hutchins et al., Proc. Natl. Acad. Sci. USA, 92: 11980-11984, 1995). IgG1 was modified in this motif by replacing Glu233-Leu-Leu235 with Pro233-Val-Ala235, a sequence from IgG2. This substitution resulted in an IgG1 mutant that lacked the ability mediated by FcγR to deplete target cells in mice (see, eg, Isaacs et al., J. Immunol., 161: 3862-3869, 1998). .

  A second portion that appears to be important for binding to both FcγR and C1q is located near the carboxy terminus of the CH2 domain of human IgG (see, eg, Duncan et al., Nature, 332: 738-740). , 1988). Of the four human IgG isotypes, only two amino acid residues show substitution within this part: Ser330 and Ser331 in IgG4 are replaced by Ala330 and Pro331 present in IgG1, IgG2 and IgG3 (FIG. 1). The presence of Ser330 does not affect binding to FcγR or C1q. Replacing Pro331 in IgG1 with Ser substantially abolished the ability of IgG1 to bind to C1q, while replacing Ser331 with Pro partially restored the complement binding activity of IgG4 (eg, Tao et al. , J. Exp. Med., 178: 661-667, 1993; Xu et al., J. Biol. Chem., 269: 3469-3474, 1994).

  In addition to the native human IgG Fc, the inventors have noted that at least three other Fc variants (vFc) can be designed and / or used for the production of the hUTI-Fc fusion proteins of the invention. I found it (Figure 1). Human IgG2 Fc does not bind to FcγR, but exhibits weak complement activity. Fcγ2 variants with the Pro331Ser mutation should have lower complement activity than native Fcγ2, but still do not bind to FcγR. IgG4 Fc is deficient in activation of the complement cascade, and its binding affinity to FcγR is about one order of magnitude less than IgG1, the most active isotype. Fcγ4 variants with Ser228Pro and Leu235Ala mutations should show minimal effector function compared to native Fcγ4. Fcγ1 mutants with Leu234Val, Leu235Ala and Pro331Ser mutations also show much lower effector function than native Fcγ1. We also find that these Fc variants, as well as native Fc from human IgG (IgG1, IgG2, IgG4) are all suitable for the preparation of hUTI-Fc fusion proteins. It is also possible to introduce other substitutions for the preparation of non-soluble Fc or native Fc without compromising the circulation half-life or causing any undesirable conformational changes.

  In the present invention, a hUTI recombinant fusion protein is produced by a cell line derived from a Chinese hamster ovary (CHO) cell line. rhUTI-Fc fusion proteins are readily purified from cell cultures using similar host cells without the inclusion of protein aggregates reported to be present in rhUTI cell cultures (see, eg, Seefeldt MB et al. al. Protein Sci., 13: 2639-50, 2004). hUTI fusion protein (hUTI-L-Fc) with an appropriate peptide linker between hUTI and Fc portion retains activity equivalent to hUTI on a molar basis when compared to hUTI for biological activity in vitro To do. The Fc portion of these molecules is known to exhibit a long serum half-life, which can reduce the frequency of administration of this long-acting drug and can be better managed. And / or more desirable serum levels are maintained.

  The present invention has many advantages, which will be particularly apparent to those skilled in the art, as disclosed and discussed below. Compared to rhUTI in mammalian cell culture, a simpler purification protocol and higher production yield of rhUTI-L-Fc can substantially reduce manufacturing costs. Different and / or higher activity and different and / or prolonged presence of hUTI-L-Fc fusion proteins in serum can result in different or lower doses, as well as different or less frequent injections. Less variation in serum drug concentration also means improved safety and tolerability. Therefore, hUTI-L-Fc fusion proteins comprising non-soluble Fc variants or native Fc contribute significantly to the management of various conditions associated with acute pancreatitis, disseminated intravascular coagulation (DIC) and shock.

Summary of the Invention One aspect of the present invention relates to hUTI-L-Fc fusion proteins. This hUTI-L-Fc fusion protein comprises hUTI, a peptide linker (indicated by L) and a human natural Fc or IgG Fc variant (both indicated by Fc). It is preferred to use a mobile peptide linker having a length of about 20 or less, more preferably about 2-20 amino acids, and the mobile peptide linker is selected from the group consisting of glycine, serine, alanine and threonine. Contains one or more amino acids. IgG Fc variants are non-soluble and contain amino acid mutations compared to native IgG Fc.

  Another aspect of the invention is that the human Ig Fc comprises the hinge, CH2 and CH3 domains of human IgG, such as human IgG1, IgG2 and IgG4. Preferably, the CH2 domain comprises amino acid mutations at positions 228, 234, 235 and 331 (defined by the EU numbering system). These amino acid mutations are thought to act to attenuate the effector function of Fc.

  In yet another aspect of the invention, methods for producing or producing such recombinant fusion proteins from mammalian cell lines, such as cell lines derived from CHO, are disclosed. The transfected cell line is grown under conditions where the recombinant fusion protein is expressed and secreted into the growth medium in an amount of 50 μg per million cells, preferably over 100 μg, over a 24 hour period. These rhUTI-L-Fc fusion proteins are characterized by a high biological activity in vitro and an extended serum half-life without undesirable side effects, on a molar basis, compared to rhUTI. This results in improved pharmacokinetics and pharmacodynamics, thus requiring lower doses and fewer injections to achieve similar efficacy.

A further aspect of the invention provides a method for producing a recombinant fusion protein comprising hUTI, a mobile peptide linker and a human IgG Fc (selected from the group consisting of variant and native forms), said method comprising: (A) generating a cell line derived from CHO; (b) the recombinant fusion protein in an amount of 50 μg per million (10 ⁶ ) cells, preferably greater than 100 μg, over a 24 hour period. Growing the cell line under conditions expressed and secreted into the growth medium; and (c) purifying the expressed protein from step b), wherein the recombinant fusion protein is similar to hUTI on a molar basis. Characterized by and indicating as much higher or higher biological activity. In this case, preferably a mobile peptide linker comprising no more than about 20, but preferably no less than 2, more preferably 20 to 2, amino acids is a hUTI and human IgG Fc (mutated and native forms). And the mobile peptide linker comprises two or more amino acids selected from the group consisting of glycine, serine, alanine and threonine; and human IgG Fc (naturally occurring) Either form or variant) comprises a hinge domain, a CH2 domain and a CH3 domain. The variant of Fc is selected from the group consisting of human IgG2, including the Pro331Ser mutation, human IgG4, including the Ser228Pro and Leu235Ala mutations, and human IgG1, including the Leu234Val, Leu235Ala, and Pro331Ser mutations.

FIG. 1 shows the amino acid sequence alignment of the hinge and CH2 regions of human IgG1, IgG2, IgG4 and variants thereof. The three parts of amino acids 228, 234-237 and 330-331 are compared. Mutant amino acid mutations are shown in bold italics. The EU numbering system is used for amino acid residues. FIG. 2 shows the nucleotide sequence (SEQ ID NO: 1) and deduced amino acid sequence (SEQ ID NO: 2) of hUTI-L-vFcγ2. The peptide of amino acid residues 1-19 is a hUTI leader peptide. The mature protein includes hUTI (amino acid residues 20-162), peptide linker (amino acid residues 163-178) and Fcγ2 variant (amino acid residues 179-401). FIG. 3 shows the nucleotide sequence (SEQ ID NO: 3) and deduced amino acid sequence (SEQ ID NO: 4) of hUTI-L-vFcγ4. The peptide of amino acid residues 1-19 is a hUTI leader peptide. The mature protein includes hUTI (amino acid residues 20-162), peptide linker (amino acid residues 163-178) and Fcγ4 variant (amino acid residues 179-407). FIG. 4 shows the nucleotide sequence (SEQ ID NO: 5) and deduced amino acid sequence (SEQ ID NO: 6) of hUTI-L-vFcγ1. The peptide of amino acid residues 1-19 is a hUTI leader peptide. The mature protein includes hUTI (amino acid residues 20-162), peptide linker (amino acid residues 163-178) and Fcγ1 variant (amino acid residues 179-405). FIG. 5 shows a diagram of the expression plasmid PCDNA3-hUTI-L-vFcγ. The plasmid, as indicated, is 9,063 bp long and contains the hCMV promoter-enhancer, fusion protein gene, DHFR, neomycin selection gene, and the like. FIG. 6 shows cell growth and cumulative protein concentration in shake flasks using a recombinant cell line secreting hUTI-L-vFcγ2 fusion protein. The final protein concentration approached 3 g / L on day 15. FIG. 7 shows an in vitro biological assay using hUTI (Techpool) purified from human urine as a trypsin inhibitor or rhUTI-L-vFcγ2 fusion protein from CHO cell supernatant. The IC50 for hUTI is 81.5 ± 6.0 nM and the IC50 for rhUTI-L-vFcγ2 is 70.9 ± 1.5 nM. FIG. 8 shows the biological activity of rhUTI-L-vFc and hUTI in a mouse acute pancreatitis model induced by cerulein. Compared to the control (Group A), hUTI had no effect on increasing serum amylase levels at the 1 mg / kg dose (Group B). At a much higher dose of 40 mg / kg (or 1 μmol / kg) hUTI showed an 8% inhibitory effect on serum amylase increase (Group C). At 3 mg / kg (or 0.027 μmol / kg), hUTI-L-vFc showed a 26% inhibitory effect on serum amylase increase (p <0.005, group D). At 30 mg / kg, inhibition continued to be much higher at 53% (p <0.001, group E). The dose of rhUTI-L-vFc in group D was 30 times less than that in group C, but the inhibitory effect on serum amylase levels was significantly better. FIG. 9 shows hUTI (Techpool) purified from human urine and purified rhUTI-L-vFcγ2 fusion protein on 10% reduced SDS-PAGE.

Detailed Description of the Invention The present invention relates to a human urinary trypsin inhibitor (hUTI) -L-Fc fusion protein comprising hUTI, a peptide linker (L) and a human IgG Fc (either native or variant). In this fusion protein, the flexible peptide linker comprises about 20 amino acids or less; and the Fc is selected from natural human IgG Fc and Fc variants (vFc). More preferably, a mobile peptide linker (L) 2 to about 20 amino acids in length is used, and the mobile peptide linker consists of two or more amino acids selected from the group consisting of glycine, serine, alanine and threonine. Or include. IgG Fc variants are non-soluble and contain amino acid mutations compared to native IgG Fc.

  The present invention has many advantages. High activity and / or prolonged presence of hUTI-L-Fc fusion protein in serum can result in lower doses and less frequent injections. Less variation in serum drug concentration also means improved safety and tolerability. Therefore, hUTI-L-Fc fusion proteins comprising non-soluble Fc variants or native Fc contribute significantly to the management of various conditions associated with acute pancreatitis, disseminated intravascular coagulation (DIC) and shock.

  In addition to native human IgG Fc, we used at least three Fc variants (vFc) for the production of hUTI-L-Fc fusion proteins and incorporated into hUTI-L-Fc fusion proteins, We have found that and / or can be engineered to produce hUTI-L-Fc fusion proteins (FIG. 1). Human IgG2 Fc does not bind to FcγR, but exhibits weak complement activity. Fcγ2 variants with the Pro331Ser mutation should have weaker complement activity than native Fcγ2, but still do not bind to FcγR. IgG4 Fc is insufficient to activate the complement cascade, and its binding affinity to FcγR is about one digit lower than IgG1, the most active isotype. Fcγ4 variants with Ser228Pro and Leu235Ala mutations should show minimal effector function compared to native Fcγ4. Fcγ1 mutants with Leu234Val, Leu235Ala and Pro331Ser mutations also show much lower effector function than native Fcγ1. We find that these Fc variants, as well as native Fc from human IgG (IgG1, IgG2, IgG4) are all suitable for the preparation of UTI fusion proteins. It is also possible to introduce other substitutions for the preparation of non-soluble Fc or native Fc without compromising the circulation half-life or causing any undesirable conformational changes.

  In accordance with the present invention, it has been discovered that the added peptide linker that exists between hUTI and human IgG Fc contributes to the in vitro biological activity of hUTI-L-Fc molecules in two ways: ( 1) Keep the Fc region away from the protease binding site on hUTI and / or (2) keep one UTI domain away from the other UTI domain. In this way, it is possible that both UTI domains can interact independently with the protease. In the present invention, a mobile peptide linker (L) having an amino acid length of about 20 or less is preferred. More preferably, the peptide linker (L) should have a length of at least 2 amino acids. It is even more preferred to use a peptide linker comprising two or more amino acids selected from the group consisting of glycine, serine, alanine and threonine.

  Also according to the present invention, the hUTI recombinant fusion protein was produced by a cell line derived from a Chinese hamster ovary (CHO) cell line. Recombinant hUTI-L-Fc appeared to be much more effective than hUTI on a molar basis when compared to hUTI for in vivo biological activity in a mouse acute pancreatitis disease model. In rats, the half-life of recombinant hUTI-L-Fc was observed to be much longer than hUTI. By producing this long acting pharmaceutical, it may be possible to provide options to reduce dosing frequency and / or manage dosing frequency and maintain high serum levels.

  For the present invention, a method is disclosed for producing or producing the recombinant fusion proteins disclosed herein from mammalian cell lines, such as the CHO derived cell lines discussed above. The transfected cell line is subjected to conditions under which the recombinant fusion protein is expressed and secreted into its growth medium in an amount greater than 50 μg per million cells, preferably greater than 100 μg, over a 24 hour period. Proliferate. These rhUTI-L-Fc fusion proteins are compared to hUTI in serum on a molar basis with good and / or high biological activity in vitro and without many undesirable side effects. Characterized by and exhibiting a half-life, which results in improved pharmacokinetics and pharmacodynamics. Thus, smaller doses and fewer injection times are required to achieve similar efficacy. Alternatively or in combination, different dosing schedules and / or different methods of injection management may be possible.

A further aspect of the invention provides a method for producing a recombinant fusion protein comprising hUTI, a mobile peptide linker and a human IgG Fc (including mutant and native forms), said method comprising (a) CHO (B) recombinant recombinant protein is expressed in its growth medium in an amount of 50 μg per million (10 ⁶ ) cells, preferably greater than 100 μg, over a 24 hour period; Growing the cell line under secreted conditions; and (c) purifying the expressed protein from step b), wherein the recombinant fusion protein has a biological activity as high as hUTI on a molar basis. And is characterized by this. In this case, preferably a mobile peptide linker comprising no more than about 20 but no less than 2 amino acids is present between rhUTI and human IgG Fc (including mutant and native forms). And the mobile peptide linker comprises two or more amino acids selected from the group consisting of glycine, serine, alanine and threonine; and human IgG Fc (either native or variant) comprises a hinge domain, Includes CH2 and CH3 domains. The variant form of Fc is selected from the group consisting of human IgG2, including the Pro331Ser mutation, human IgG4, including the Ser228Pro and Leu235Ala mutations, and human IgG1, including the Leu234Val, Leu235Ala, and Pro331Ser mutations.

Example 1. Preparation of Gene Encoding hUTI-L-vFcγ2 Fusion Protein A gene encoding a human UTI leader peptide and a mature protein was newly synthesized. The obtained DNA fragment of about 500 bp was inserted into the EcoRV restriction enzyme site of a holding vector such as pUC57 to obtain a phUTI plasmid. The sequence of the hUTI gene was confirmed by DNA sequencing.

  A gene encoding a linker peptide and an Fc variant region of human IgG2 (Fcγ2 containing Pro331Ser mutation) was synthesized. The obtained Fcγ2 DNA fragment is a sequence of an IgG2 hinge domain, a CH2 domain, and a CH3 domain containing a linker peptide and an Fcγ2 Pro331Ser mutant (vFcγ2) (Pro at position 331 of Fcγ2 was replaced with Ser) Included. The linker peptide contained a sequence encoding a 16 amino acid Gly-Ser peptide linker. The obtained DNA fragment of about 700 bp was inserted into BamHI and EcoRI sites of a holding vector such as pUC19 to obtain a pL-vFcγ2 plasmid. The sequence of the gene was confirmed by DNA sequencing.

  To prepare the hUTI-L-vFcγ2 fusion gene, the hUTI fragment was excised from the phUTI plasmid using SpeI and BamHI and purified by agarose gel electrophoresis. The purified fragment was then inserted into the 5 'end of the peptide linker in the pL-vFcγ2 plasmid to obtain the phUTI-L-vFcγ2 plasmid. The fusion gene includes hUTI, Gly-Ser peptide linker and Fcγ2 mutant gene.

  The presence of a peptide linker, preferably a mobile linker, between (and chemically bound to) the hUTI and Fc moieties increases the mobility of the hUTI domain and enhances its biological activity. In the present invention, a peptide linker having an amino acid length of about 20 or less is preferred. One amino acid is also within the scope of the invention, but preferably has a mobile peptide linker of about 20 to about 2 amino acids in length. Peptide linkers comprising two or more amino acids selected from the group consisting of glycine, serine, alanine and threonine can preferably be used. Examples of peptide linkers include Gly-Ser peptide building blocks such as GlyGlyGlyGlySer. FIG. 2 shows a fusion gene comprising a sequence encoding hUTI, a 16 amino acid peptide linker (GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer) and Fcγ2 Pro331Ser variant, and its corresponding amino acid sequence. FIG. 3 shows the fusion gene and corresponding amino acid sequence for hUTI-L-vFcγ4 (Fcγ4 with Ser228Pro and Leu235Ala mutations). FIG. 4 shows the fusion gene and corresponding amino acid sequence for hUTI-L-vFcγ1 (Fcγ1 with Leu234Val, Leu235Ala and Pro331Ser mutations).

Example 2 Construction of an expression vector for hUTI-Fc The complete gene encoding the hUTI-L-vFcγ fusion protein was then inserted into the HindIII and EcoRI sites of mammalian expression vectors such as pcDNA3 (Invitrogen). As shown in FIG. 5, the final expression vector plasmid pCDNA3-hUTI-L-vFcγ contains a cytomegalovirus early gene promoter-enhancer that is required for high level expression in mammalian cells. The plasmid also contains a selectable marker for conferring ampicillin resistance in bacteria and G418 resistance in mammalian cells. Furthermore, this expression vector is a dihydrofolate reductase that allows simultaneous amplification of the hUTI-L-vFcγ fusion gene and the DHFR gene in the presence of methotrexate (MTX) when the host cell is deficient in DHFR gene expression. DHFR) gene (see, for example, US Pat. No. 4,399,216).

Example 3 Expression of the fusion protein in transfected cell lines Expression of the fusion protein was achieved by transfecting a mammalian host cell line using the pCDNA3-hUTI-L-vFcγ expression vector plasmid. Preferred host cell lines for stable high level expression are CHO cells deficient in DHFR enzyme (see, eg, US Pat. No. 4,818,679). A preferred transfection method is electroporation. Other methods including calcium phosphate coprecipitation, Lipofectamine 2000 can also be used. For electroporation, 20 μg of plasmid DNA chained with PvuI was used with a Gene Pulser electroporator (Bio-Rad Laboratories, Hercules, Calif.) Set to an electric field of 250 V and a capacitance of 960 μFd. And added to 2-5 × 10 ⁷ cells in the cuvette. Two days after transfection, the medium was replaced with growth medium containing 0.8 mg / ml G418. Transfectants resistant to the selected drug were tested for secretion of the fusion protein using an anti-human IgG Fc ELISA. Quantification of the expressed fusion protein was also performed by anti-hUTI ELISA. Wells producing high levels of Fc fusion protein were subcloned by limiting dilution on 96-well tissue culture plates.

  To achieve higher levels of fusion protein expression, co-amplification was performed by using a gene for DHFR that can be inhibited by MTX drugs. In a growth medium containing increasing concentrations of MTX, the gene for the transfected fusion protein was co-amplified with the DHFR gene. Transfectants capable of growing in medium containing up to 1 μg / ml MTX were subcloned again by limiting dilution. Subcloned cell lines were further analyzed by measuring secretion rate. Several cell lines that produced secretion rate levels in excess of 100 μg per million cells per 24 hours were adapted to suspension culture using serum-free growth medium. One cell line was grown in 100 ml using a shake flask for 15 days. FIG. 6 shows the cumulative concentration of fusion protein of about 3 g / L. On days 5 to 10, the maximum viable cell density remained at about 6 million cells per ml. Under these conditions, the secretion rate is calculated to be about 50 μg per million cells per 24 hours. Conditioned medium was used for purification of the fusion protein.

Example 4 Purification and characterization of the fusion protein The conditioned medium containing the fusion protein was titrated with 1N NaOH to pH 7-8 and filtered through a 0.45 micron nitrocellulose filter. The filtrate was loaded onto a ProsepA column equilibrated in phosphate buffered saline (PBS). After the fusion protein was bound to ProsepA, the flow-through fraction was discarded. The column was washed with PBS until the OD at 280 nm was less than 0.01. The bound fusion protein was then eluted with 0.1 M citrate buffer at pH 3.75. After neutralization with 0.4 volume of 1M K ₂ HPO ₄ , fractions containing purified protein were pooled and dialyzed against PBS. The solution was then filtered through a 0.22 micron nitrocellulose filter and stored at -70 ° C. As shown by 10% reducing SDS-PAGE analysis (FIG. 9), the molecular weight of the purified hUTI-L-vFc protein was estimated to be about 55 kD. Under similar conditions, the molecular weight of hUTI (Techpool) purified from human urine appeared to be 38 kD. The fusion protein was quantified by BCA protein assay using BSA as a standard.

Example 5 FIG. In vitro biological activity assay The biological activity of UTI in the supernatant of the transfectants or purified protein was assayed by determining the residual trypsin activity according to standard procedures (eg Kakade et al Cereal Chem., 46: 518-526, 1969). As substrates BAPNA (N α _- benzoyl -L- arginine 4-nitroanilide hydrochloride, Sigma-Aldrich) was performed modified by the use of porcine trypsin (Sigma-Aldrich) and as an enzyme. Reactions containing 20 μl of diluted hUTI protein, purified hUTI-L-vFcγ, or supernatant of the transfectant, 5 μl trypsin (100 mg / ml solution in 1 mM HCl containing 2.94 g / L CaCl ₂ ) The mixture was adjusted to a volume of 125 μl with 0.2 M triethanolamine buffer. After incubating the mixture for 20 minutes at 37 ° C. with shaking, 5 μl of BAPNA (25 mg dissolved in a minimum volume of DMSO and brought to a final concentration of 10 mg / ml using 0.2 M triethanolamine buffer. Adjusted) was added to the mixture and the incubation continued for another 20 minutes with shaking at 37 ° C. The reaction was then terminated by the addition of 30 μl 30% (v / v) glacial acetic acid. Blank and trypsin controls were performed simultaneously. Absorbance at 405 nm was recorded. FIG. 7 shows the normalized activity of hUTI (Techpool) or purified rhUTI-L-vFcγ fusion protein versus the molar (nM) concentration of hUTI (Techpool) or purified rhUTI-L-vFcγ fusion protein. The concentration of is shown. Under these conditions, the IC50 was estimated to be 81.5 ± 6.0 nM for hUTI and 70.9 ± 1.5 nM for the purified rhUTI-L-vFcγ2 fusion protein.

Example 6 In vivo biological activity in mouse disease models In vivo biological activity of purified rhUTI-L-vFc protein is used in a widely accepted acute pancreatitis mouse model induced with cerulein And tested. Twenty-four hours after injection with cerulein, the mice develop biochemical, histological, and immunohistochemical evidence of acute pancreatitis. Serum biochemical markers such as amylase, lipase, TNF-α and IL-1β were elevated. Using immunohistochemical staining methods, it was found that levels of leukocyte adhesion molecules, vascular endothelial growth factor and apoptosis-related proteins were increased in these mice (eg, Ding et al, Hepatobiliary Pancreat. Dis. Int ., 9: 645-650, 2010; Galuppo et al, Br J. Pharmacol., 162: 1186-1201, 2011). In our experiments, serum amylase activity was followed as an indicator of the severity of acute pancreatitis.

  Thirty 8-week-old male C57BL / 6 mice (20-22 g) (SLAC Laboratory, Shanghai) were randomly divided into 5 groups of 6 per group. After the mice were fasted for 15 hours, each group was injected intraperitoneally with 50 μg / kg of celluline 6 times at 1 hour intervals. In mice receiving serulein injection as above, amylase concentration increased rapidly after the last injection and peaked after 3 hours. Thereafter, the level slowly decreased and returned to baseline after 24 hours. This observation indicated that 1 hour after the last cellulin injection was the best time to assess the efficacy of treatment with hUTI or rhUTI-L-vFc.

  Following the first and fifth injections of cerulein, mice were injected through their tail vein with hUTI (Techpool) or rhUTI-L-vFc purified from human urine. As shown in FIG. 8, mice in groups B and C are treated with 1 mg / kg and 40 mg / kg hUTI, respectively; mice in groups D and E are treated with 3 mg / kg and 30 mg, respectively. Treated with / kg rhUTI-L-vFc. Group A mice served as controls that received an equal volume of saline. After the last injection of cerulein, 200 μl blood samples were collected from mice through their orbits at various time points. Samples were centrifuged at 6000 rpm for 10 minutes and serum was stored at -70 ° C until the assay was performed. The assay was performed on a Hitachi 7060 Automatic Chemistry Analyzer according to the manufacturer's instructions. Amylase concentration was expressed in international units per liter.

  Compared to the control (Group A), hUTI had no effect on increasing serum amylase concentration at the 1 mg / kg dose (Group B). At a much higher dose of 40 mg / kg, hUTI showed an 8% inhibitory effect on the increase in serum amylase (Group C). However, treatment with hUTI-L-vFc significantly reduced serum amylase levels compared to controls (Group A). hUTI-L-vFc showed 26% inhibitory effect on serum amylase increase at 3 mg / kg (p <0.005). At 30 mg / kg, inhibition appeared to be much more effective at 53% (p <0.001). Molar concentrations were further calculated by using the estimated molecular weights of monomeric rhUTI-L-vFc and hUTI as 55 kD and 38 kD, respectively (FIG. 9). Therefore, 40 mg / kg or 1 μmol / kg hUTI has an 8% inhibitory effect, while 3 mg / kg or 0.027 μmol / kg rhUTI-L-vFc is against increasing serum amylase concentration. It had an inhibitory effect of 26%. On a molar basis, the dosage of rhUTI-L-vFc was 30 times less than hUTI, however, inhibition against serum amylase was much more effective. These results confirmed that rhUTI-L-vFc is more effective than hUTI in treating acute pancreatitis in these mice.

Example 7 Serum half-life in rats SD rats (SLAC Laboratory, Shanghai) were injected with a single dose (2 mg / kg) of purified hUTI-L-vFc through the tail vein. Blood samples were collected at various time intervals and centrifuged at 6000 rpm for 10 minutes. Plasma supernatant was stored at -70 ° C until the assay was performed. Serum concentrations were tested by ELISA using polyclonal goat anti-human IgG Fc antibody and horseradish peroxidase labeled mouse anti-hUTI antibody. The half-life of hUTI-L-vFc was estimated to be 910 minutes, compared to 182 minutes for hUTI. Therefore, the half-life of hUTI-L-vFc is significantly prolonged in rats compared to hUTI.

Claims (13)

  A hUTI-L-Fc fusion protein comprising human urinary trypsin inhibitor (hUTI), peptide linker (L) and human IgG Fc, wherein the mobile peptide linker comprises about 20 amino acids or less in length; and The hUTI-L-Fc fusion protein, wherein the human IgG Fc is selected from the group consisting of native IgG Fc and Fc variants (vFc).   2. The fusion protein of claim 1, wherein the IgG Fc variant is non-soluble and contains amino acid mutations compared to native IgG Fc.   The human IgG Fc comprises a human IgG hinge domain, CH2 domain and CH3 domain selected from the group consisting of human IgG1, human IgG2 and human IgG4, and the CH2 domain is at positions 228, 234, 235 and 331 The fusion protein of claim 2 comprising an amino acid mutation in   The IgG Fc variant is non-soluble; the human Ig Fc comprises a human IgG hinge domain, CH2 domain and CH3 domain selected from the group consisting of human IgG1, human IgG2 and human IgG4; and the CH2 domain The fusion protein of claim 1 comprising amino acid mutations at positions 228, 234, 235 and 331.   The fusion protein of claim 1, wherein the mobile peptide linker is present between hUTI and human IgG Fc, and the mobile peptide linker comprises 2 to 20 amino acids in length.   The mobile peptide linker is present between hUTI and human IgG Fc, and the mobile peptide linker comprises 2-20 amino acids in length, and the amino acids are glycine, serine, alanine and threonine The fusion protein of claim 1 selected from the group consisting of:   A hUTI-L-Fc fusion protein comprising human urinary trypsin inhibitor (hUTI), peptide linker (L) and human IgG Fc, wherein the mobile peptide linker is present between hUTI and human IgG Fc; And comprising 2 to 20 amino acids in length and selected from the group consisting of glycine, serine, alanine and threonine; Fc is a native IgG Fc and a non-soluble, amino acid mutation compared to native IgG Fc A human Ig Fc comprises a hinge domain, a CH2 domain and a CH3 domain; and a human IgG Fc comprises a human IgG2, Ser228Pro and Leu235Ala mutation comprising a Pro331Ser mutation. Human IgG4 containing, as well as Le 234Val, is selected from the group consisting of human IgG1 comprising Leu235Ala and Pro331Ser mutation, the hUTI-L-Fc fusion protein. A method for producing a recombinant fusion protein comprising hUTI, a mobile peptide linker, and a human IgG Fc comprising a hinge domain, a CH2 domain and a CH3 domain, comprising: (a) a cell line derived from CHO (B) a cell line under conditions where the recombinant fusion protein is expressed and secreted in its growth medium in an amount greater than 50 μg per million (10 ⁶ ) cells over a 24 hour period And c) purifying the expressed protein from step b). The cell line is grown under conditions such that in step (b) the recombinant fusion protein is expressed and secreted in its growth medium in an amount greater than 50 μg per million (10 ⁶ ) cells over a 24 hour period. The method of claim 8.   Human IgG Fc is a non-soluble human Fc selected from the group consisting of human IgG2, including the Pro331Ser mutation, human IgG4 including the Ser228Pro and Leu235Ala mutations, and human IgG1 including the Leu234Val, Leu235Ala and Pro331Ser mutations 10. The method of claim 9, wherein the method is selected from the group consisting of variant Fc.   A human IgG Fc from a native Fc and a variant Fc selected from the group consisting of human IgG2, including the Pro331Ser mutation, human IgG4 including the Ser228Pro and Leu235Ala mutations, and human IgG1 including the Leu234Val, Leu235Ala and Pro331Ser mutations 9. The method of claim 8, wherein the method is selected from the group consisting of:   9. The method of claim 8, wherein the mobile peptide linker comprises 2 to 20 amino acids selected from the group consisting of glycine, serine, alanine and threonine. A cell line is grown under conditions in which the recombinant fusion protein is expressed and secreted in its growth medium in an amount greater than 50 μg per million (10 ⁶ ) cells during a 24 hour period; and The recombinant fusion protein is characterized by and exhibits biological activity similar to hUTI on a molar basis; a mobile peptide linker is present between hUTI and IgG Fc; human IgG Fc is native Fc, Selected from the group consisting of a human IgG2, including the Pro331Ser mutation, a human IgG4 including the Ser228Pro and Leu235Ala mutations, and a variant Fc selected from the group consisting of human IgG1 including the Leu234Val, Leu235Ala and Pro331Ser mutations; and said mobile Sex peptide linkers 9. The method of claim 8, comprising 2-20 amino acids selected from the group consisting of syn, serine, alanine and threonine.