Patent Application
20220267412
Human serpin B1 is a member of the serine proteinase inhibitor (serpin) superfamily that has anti-inflammatory properties. The anti-inflammatory properties are, in part, attributable to its ability to inhibit pro-inflammatory neutrophil serine proteases (NSP's) via the reactive site loop (RSL) and the ability to limit the self-association and spontaneous activation of pro-caspases via a caspase recruitment domain binding motif (CBM) located C-terminal to the RSL (Cooley, et al., 2001; Choi et al., 2019) Human serpin B1 can inhibit the NSP's cathepsin G and elastase through efficient reactions at 2 overlapping reactive sites: Phe-343 and Cys-344.
In some aspects, this disclosure provides a SERPIN B1 variant polypeptide that comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, and the SERPIN B1 variant polypeptide possesses neutrophil or pancreatic elastase inhibitory activity and the elastate inhibition activity of the SERPIN B1 variant polypeptide is resistant to oxidation by free radicals. The free radicals may be reactive oxygen species, or reactive nitrogen species, or both. In some embodiments, the SERPIN B1 variant polypeptide comprises an amino acid substitution at residue 344 as compared to SEQ ID NO: 1. In some embodiments, the amino acid substitution is selected from the group consisting of C344A, C344V, and C344G.
In some embodiments, the SERPIN B1 variant polypeptide disclosed herein is fused to an Fc portion of an IgG, a single chain variable fragment (scFv) of an antibody. In some embodiments, the SERPIN B1 variant polypeptide is pegylated.
Also provided herein is a polynucleotide encoding any of the SERPIN B1 variant polypeptides disclosed in this disclosure.
Also provided herein is a pharmaceutical composition comprising any of the SERPIN B1 polypeptides disclosed in this application and a pharmaceutically acceptable excipient. In some embodiments, a pharmaceutical composition comprises a SERPIN B1 polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and a reducing agent that prevents oxidation of cysteine 344, wherein the polypeptide is capable of inhibiting neutrophil or pancreatic elastase. In some embodiments, the reducing agent is N-acetylcysteine (NAC).
Also provided herein is a method of treating a patient having a disease that is associated with increased production of free radicals as compared to a normal individual or increased exposure to free radicals in environmental sources. The method comprises administering a SERPIN B1 variant polypeptide, wherein the SERPIN B1 variant polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1, wherein the SERPIN variant polypeptide comprises an amino acid substitution at residue 344, as compared to the native protein sequence of SEQ ID NO: 1; said SERPIN B1 variant polypeptide is capable of inhibiting the serine protease activity of a neutrophil or pancreatic elastase and is resistant to oxidation by a free radical. In some embodiments, the free radical is a reactive oxygen species, a reactive nitrogen species, or both. In some embodiments, the SERPIN variant polypeptide comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 2-4.
Also provided herein is a method of treating a patient having a disease or a genetic condition that is associated with the increased production of free radicals as compared to a normal individual or increased exposure to free radicals in environmental sources, wherein the method comprises administering the pharmaceutical composition of comprising the SERPIN B1 variant polypeptide disclosed herein. Also provided is a method of treating a patient having a disease or a genetic condition that is associated with increased production of free radicals as compared to a normal individual or an increased exposure to free radicals in environmental sources, wherein the method comprises administering the pharmaceutical composition of a SERPIN B1 polypeptide comprising a sequence that is the wild type SERPIN B1 (SEQ ID NO: 1) or a sequence that is at least 90% identical to the wild type SERPIN B1 polypeptide (SEQ ID NO: 1), and a reducing agent disclosed herein.
In some embodiments, the disease or genetic condition is associated with exposure to free radicals present in the environment (e.g. cigarette smoke, vape device emissions) or the increased production of free radicals by enzymes present in innate immune cells, mucosal cells, or glandular cells as compared to a normal individual In some embodiments, the diseases are selected from groups of infectious, autoimmune, respiratory, metabolic, cardiovascular, neurodegenerative or oncology diseases. Infectious diseases include, but are not limited to, pulmonary or systemic diseases such as acute lung injury (ALI), acute respiratory distress (ARDS), pneumonia, bronchiolitis, systemic coagulopathies or hemorrhagic diseases caused by, but not limited to, respiratory syncytial viruses, influenza viruses, coronaviruses, ebola viruses, Pseudomonas aeruginosa and other opportunistic pathogens. Autoimmune diseases include, but are not limited to, type 1 diabetes, rheumatoid arthritis, psoriasis, multiple sclerosis and sterile autoinflammatory diseases (SAID's) that have underlying genetic mutation(s) predisposing patients to recurrent bouts of episodic inflammation. Respiratory diseases include, but are not limited to, allergic asthma, smokers' emphysema, COPD and idiopathic pulmonary fibrosis (IPF). Metabolic diseases include, but are not limited to, type 2 diabetes, insulin resistance, dyslipidemia and cataract formation. Cardiovascular diseases include but are not limited to, atherosclerosis and hypertension. Neurodegenerative diseases include, but are not limited to, Parkinson's and Alzheimer's. Oncology diseases include, but are not limited to, colorectal, pancreatic, prostate, breast, lung and bladder cancers
The oxidation-resistant SERPIN B1 variant polypeptide or composition thereof disclosed herein may be administered by inhalation, intra-tracheally, topically or by injection subcutaneously, intravenously, or intraperitoneally. In some embodiments, the SERPIN B1 variant polypeptide or the wild type SERPIN B1 (SEQ ID NO: 1) is administered at a dose of 0.01 mg to 1000 mg per kg of patient's mass (i.e., 0.01 mg/kg to 1000 mg/kg). In some embodiments, the SERPIN B1 variant polypeptide or the wild type SERPIN B1 is administered in combination with a reducing agent, where the reducing agent is administered in an amount that is sufficient to prevent the oxidation of the C344 of the wild type SERPIN B1 or SERPIN B1 variant polypeptide. In some embodiments, the reducing agent is administered in at a dose of 0.01-100 mg per kilogram of the patient's mass (i.e., 0.01-100 mg/kg).
Also provided herein is a method of producing a wild type SERPIN B1 or a variant polypeptide thereof, the method comprising: expressing the polynucleotide encoding a wild type SERPIN B1 or a variant polypeptide thereof in S. cerevisiae, wherein the SERPIN B1 variant polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1, wherein the SERPIN variant polypeptide comprises an amino acid substitution at residue 344, as compared to the native protein sequence of SEQ ID NO: 1; wherein the SERPIN B1 variant polypeptide is capable of inhibiting the serine protease activity of neutrophil or pancreatic elastase; and wherein the SERPIN B1 variant polypeptide is resistant to oxidation by free radicals. In some embodiments, the S. cerevisiae is protease-deficient.
In some embodiments, the method of expressing the polynucleotide is by introducing a Yeast episomal expression plasmid (Yep) into the S. cerevisiae. In some embodiments, the method of embodiment 19, wherein the polynucleotide is linked to a yeast promoter. In some embodiments, the yeast promoter is an ADH2 promoter. In some embodiments, the polynucleotide is codon optimized for expression in yeast.
In some embodiments, the Serpin B1 variant polypeptide is fused to a Fc portion of an IgG, a single chain variable fragment (scFv) of an antibody, or wherein the Serpin B1 variant polypeptide is pegylated.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.
All numerical designations, e.g., pH, temperature, time, concentration, amounts, a molecular weight, including ranges, are approximations which are varied (+) or (−) by increments of 0.1 or 1.0, where appropriate. It is to be understood, although not always explicitly stated, that all numerical designations may be preceded by the term “about.” It is also to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.
“Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
The term “comprising” is intended to mean that the compositions and methods include the recited elements, but do not exclude others. “Consisting essentially of” when used to define compositions and methods, refers to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. “Consisting of” shall mean excluding more than trace amounts of other ingredients and substantial method steps recited. Embodiments defined by each of these transition terms are within the scope of this invention.
The term “rhsB1,” disclosed herein refers to the native human SERPIN B1 polypeptide (SEQ ID NO: 1) that is produced in non-human host cells.
The term “SERPIN B1 polypeptide,” or “SERPIN B1,” refers to a native (also referred to as “wild type”) SERPIN B1 polypeptide having the sequence of SEQ ID NO: 1, or a variant thereof (i.e., a SERPIN B1 variant polypeptide).
The terms “polynucleotide”, “nucleic acid” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three dimensional structure and may perform any function, known or unknown. The following are non limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double and single stranded molecules. Unless otherwise specified or required, any embodiment of this invention that is a polynucleotide encompasses both the double stranded form and each of two complementary single stranded forms known or predicted to make up the double stranded form.
A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule.
The term “percent identity” refers to sequence identity between two peptides or between two nucleic acid molecules. Percent identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are identical at that position. As used herein, the phrase “homologous” or “variant” nucleotide sequence,” or “homologous” or “variant” amino acid sequence refers to sequences characterized by identity, at the nucleotide level or amino acid level, of at least a specified percentage. Homologous nucleotide sequences include those sequences coding for naturally occurring allelic variants and mutations of the nucleotide sequences set forth herein. Homologous nucleotide sequences include nucleotide sequences encoding for a protein of a mammalian species other than humans. Homologous amino acid sequences include those amino acid sequences which contain conservative amino acid substitutions and which polypeptides have the same binding and/or activity. In some embodiments, a homologous nucleotide or amino acid sequence has at least 60% or greater, for example at least 70%, or at least 80%, at least 85% or greater, with a comparator sequence. In some embodiments, a homologous nucleotide or amino acid sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with a comparator sequence. In some embodiments, a homologous amino acid sequence has no more than 15, nor more than 10, nor more than 5 or no more than 3 conservative amino acid substitutions. Percent identity can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).
The term “express” refers to the production of a gene product. The term “transient” when referred to expression means a polynucleotide is not incorporated into the genome of the cell.
The term “vector” refers to a non-chromosomal nucleic acid comprising an intact replicon such that the vector may be replicated when placed within a permissive cell, for example by a process of transformation. A vector may replicate in one cell type, such as bacteria, but have limited ability to replicate in another cell, such as mammalian cells. Vectors may be viral or non-viral. Exemplary non-viral vectors for delivering nucleic acid include naked DNA; DNA complexed with cationic lipids, alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles comprising DNA condensed with cationic polymers such as heterogeneous polylysine, defined-length oligopeptides, and polyethylene imine, in some cases contained in liposomes; and the use of ternary complexes comprising a virus and polylysine-DNA.
The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.
The term “normal individual,” as used herein, refers to a healthy, non-smoking individual.
The term “associated with,” with regard to the relationship between a disease or genetic condition and free radicals, refers to that the disease or genetic condition is at least in part resulted from exposure to a high level of free radicals in the environment or inside the body, or that the disease or genetic condition causes the increased production of free radicals in the body as compared to a normal individual.
The term “treating” or “treatment” covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, i.e., arresting its development; (ii) relieving a disease or disorder, i.e., causing regression of the disorder; (iii) slowing progression of the disorder; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. The term “administering” or “administration” of a monoclonal antibody or a natural killer cell to a subject includes any route of introducing or delivering the antibody or cells to perform the intended function. Administration can be carried out by any route suitable for the delivery of the cells or monoclonal antibody. Thus, delivery routes can include intravenous, intramuscular, intraperitoneal, or subcutaneous deliver.
The term “administering” includes oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. One skilled in the art will know of additional methods for administering a therapeutically effective amount of a fusion protein described herein.
The term “therapeutically effective amount” or “effective mount” includes an amount or quantity effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
As used herein, the term “substantially the same”, when referring to the enzymatic inhibition activity, for example, neutrophil elastase inhibition activity, refers to that the two measurements of inhibition activity are no more than 25%, no more than 20%, no more than 15% different, no more than 10%, no more than 8%, or no more than 5% different from each other.
Human serpin B1 (hsB1) was first identified in 1985 as a fast acting elastase inhibitor present in high concentrations in human monocytes cultured in vitro and subsequently in macrophages and neutrophils (Remold-O'Donnell et al., J. Exp. Med. 162, 2142-2155 (1985); Remold-O'Donnell et al., J. Exp. Med. 169, 1071-1086 (1989), 2). It has a molecular weight of approximately 42 kDa and is a member of the clade B branch of the serpin superfamily of proteins that do not possess classical secretion signals (Remold-O'Donnell et al., Proc. Natl. Acad. Sci., USA 89, 5635-5639 (1992)). Sequence alignments with other serpins identified Cys-344 as the putative P1 residue on the reactive site loop (RSL) responsible for the observed EIA. N-terminal sequence data derived from elastase-hsB1 complexes and the sensitivity of the proteins EIA to the alkylating agent iodoacetamide subsequently confirmed this assignment (Remold-O'Donnell et al., J. Exp. Med. 169, 1071-1086 (1989); Cooley et al. Biochemistry 40, 15762-15770 (2013)). A number of serpins have been demonstrated to utilize overlapping reactive sites to efficiently inhibit different classes of proteinases. This is also true for hsB1 which utilizes Phe-343 to inhibit chymotrypsin-like proteases including bovine chymotrypsin, cathepsin G (catG), mast cell chymase, granzyme H (GmzH) and prostate specific antigen (PSA)—in addition to Cys-344 which it also utilizes to inhibit neutrophil proteinase 3 (Cooley et al.; Wang et al., J. Immunol. 190, 1319-1330 (2013)).
Serpin B1 exhibits a number of protective anti-inflammatory roles in vivo. Recombinant hsB1 has been used prophylactically to protect rat lungs against injury mediated by pro-inflammatory cystic fibrosis airway secretions, suppress bacterial proliferation in a mouse model of P. aeruginosa lung infection and ameliorate post-operative acute lung injury in a rat model of liver transplantation (Cooley et al. (1998)). In 2007, Benerafa et al. knocked out the gene for the murine homolog (sb1a) of human sB1 and demonstrated the critical role it plays in regulating excessive inflammatory responses during bacterial infection (Benerafa et al. (2007)). In 2011 Gong et al, used the same model to demonstrate that sb1a also protected against the excessive inflammation induced by pulmonary influenza without affecting viral clearance (Gong et al., (2011)). Further studies by the groups of Benarafa and O'Donnell have revealed that sb1a protects neutrophils in the bone marrow and prevents programmed necrosis in neutrophils and monocytes via the specific inhibition of cathepsin G (Benerafa et al., (2011)). In 2012, Farley et al, discovered that sB1 can regulate the formation of neutrophil extracellular traps (NETs) induced by multiple different stimuli—a process in part dependent on the production of ROS by myeloperoxidase (MPO) (Choi et al. (2019)). In this role sb1a was observed to migrate from the cytoplasm of neutrophils into the nucleus but the mechanism driving this translocation and its nuclear target(s) are currently unknown (Farley et al. (2012)). In addition, sb1a reportedly regulates the expansion of Th17 phenotype T-cells via inhibition of cysteine cathepsins, most prominently cathepsin L (Zhao et al. (2014)). More recently, El Ouaamari et al., identified sB1 as a factor that promotes compensatory (3-cell responses to insulin resistance by inducing pancreatic β-islet cell proliferation via modulation of proteins in growth and survival pathways (El Ouaamari et al. (2016)) while Choi et al identified a CARD (caspase recruitment domain) binding motif (CBM) in Serpin B1 that is responsible for limiting the activation of inflammatory caspases 1, 4, 5 and 11 and preventing pyroptosis (Choi et al. (2019)).
Others have suggested sB1 to play a direct role in apoptosis. In 1998, Torriglia et al. partially sequenced a ubiquitous intracellular cation-independent acidic endonuclease called DNase II and found it to be homologous with the protein sequence of porcine sB1 (Torriglia et al. (1998)). Since then, this group has published a number of papers suggesting that, upon cleavage by elastase or some other protease involved in apoptosis, sB1 undergoes a structural rearrangement to unmask a latent endonuclease activity in a molecule which they call L-DNase II—a truly unique and surprising activity for a serpin if confirmed (Padron-Barthe et al. (2007)).
In this application, we disclose that the exposed physical location of Cys-344 in sB1 would render it sensitive to reactive free radical species (e.g., ROS or RNS) produced during inflammatory or redox mediated signaling events. The present application discloses that maintenance of Cys-344 in a reduced state is required for sB1 to efficiently inhibit elastase and demonstrate that oxidation of Cys-344 can produce post-translationally modified (PTM) forms of sB1 with altered protease inhibitory activities. During the early stages of inflammation and perhaps other redox mediated cell-signaling events, the control functions of SERPIN B1 may be down-regulated by certain types of free radicals allowing inflammation to proceed. The present application thus provides methods and compositions comprising a SERPIN B1 variant polypeptide, in which The SERPIN B1 variant polypeptide possesses neutrophil elastase inhibitory activity and the neutrophil elastase inhibition activity is resistant to oxidation by free radicals. In some cases, the SERPIN B1 variant polypeptide has an amino acid sequence that is at least 90% identical to SEQ ID NO: 1. These SERPIN B1 variant polypeptides can be used to treat patients having a disease or genetic condition that is associated with the increased production of free radicals in neutrophils, monocytes as compared to a normal individual.
A. The Elastase Inhibition Activity of SERPIN B1 Requries Maintaining C344-SH in a Reduced State
Here we provide evidence that the structurally exposed Cys-344 (Wang et al., J. Immunol. 190, 1319-1330 (2013), is subject to post-translational modifications (PTM's) that convert sB1 from an inhibitor to substrate of elastase which results in in the loss of elastase inhibition activity and can lead to the complete loss of all protease inhibitory activity. See Examples 3, 10 and 11. The recombinant human serpin B1 (rhsB1) produced intracellularly in yeast inhibits both elastase and chymotrypsin (EIA and CIA) but EIA is sensitive to rapid inactivation by the oxidizing agents N-chlorosuccinimide, peroxynitrite and sodium hypochlorite and free radicals generated by myeloperoxidase (
These findings confirm that hsB1 is capable of inhibiting both elastase and chymotrypsin-like proteases under conditions where maintenance of a reduced Cys-344 prevails. However, we propose that during inflammation or redox-mediated cell signaling events involving certain types of reactive oxygen species (ROS) or reactive nitrogen species (RNS), PTM's of Cys-344 may be a key event allowing inflammatory pathways to proceed via increased elastase, cathepsin G and proteinase 3 activity. The conversion of hsB1 to a cleaved inactive (R) form by elastase will completely inactivate all direct protease inhibitory activity mediated by the reactive site loop and may also disrupt other regulatory domain(s) within the proteins tertiary structure (e.g. CBM) allowing the activation/amplification of other pro-inflammatory pathways.
B. Oxidation-Resistant SERPIN B1 Variants
The disclosure provides a SERPIN B1 variant polypeptide possessing neutrophil elastase inhibition activity and the neutrophil elastase inhibitory activity is resistant to oxidation by free radicals. Neutrophil (and pancreatic) elastase inhibitory activity of the native SERPIN B1 is dependent on the reduced form of C344 (“C344 dependent elastase inhibition activity”). The C344 dependent elastase inhibitory activity of SERPIN B1 is susceptible to oxidation by free radicals because the oxidation of the C344 of the SERPIN B1 results in a significant reduction or a complete loss of elastase inhibitory activity. The term “free radical” or “radical,” or “reactive free radical species” typically refers to a molecule with an unpaired electron and capable of high reactivity. Radicals have extremely high chemical reactivity and when generated in excess or not appropriately controlled, may inflict damage upon cells. Free radicals disclosed in this disclosure refers to any cysteine reactive free radical species, e.g., a reactive free radical species that can oxidize C344 of the native SERPIN B1 (SEQ ID NO: 1) and decrease its elastase inhibitory activity. The decrease in the elastase inhibitory activity may be at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% as compared to the native SERPIN B1, the C344 of which is unoxidized. These free radicals may include, but are not limited to, those derived from oxygen (“reactive oxygen species”) or those derived from nitrogen (“reactive nitrogen species”). Oxidation of cysteine by the free radicals may produce a variety of oxidation products, including, e.g., S-nitrosocysteine, cysteine sulfenic, sulfinic and sulfonic acids, disulfides and persulfides. As a result, oxidation of cysteine by free radicals may have toxicological implications in a number of diseases, such as emphysema and cancer, and impair the body's anti-bacterial and anti-viral defenses.
Free radicals are present in, or may be induced by exogenous sources such as smoke and pollution particles and can be produced by endogenous sources in response to pathogens such as viruses or bacteria, or genetic conditions predisposing an individual to “sterile” autoinflammatory diseases (SAID's). Endogenous ROS and RNS are produced by enzymes (e.g. peroxidases and nitric oxide synthases) that are resident in, or secreted from innate immune cells (e.g. neutrophils, eosinophils, macrophages, monocytes), mucosal cells (e.g. lung airway mucosa, intestinal mucosa), and glandular cells (e.g. thyroid, mammary and salivary).
Non-limiting examples of ROS include hypochlorous acid, hypochlorite, N-chlorosuccinimide, hydrogen peroxide, and sodium hypochlorite. Non-limiting examples of RNS include nitric oxide (NO). Some agents can be both ROS and RNS, for example, peroxynitrite. Other examples of reactive free radical species include, but not limited to, those as described in Griendling et al. (2016), Measurement of Reactive Oxygen Species, Reactive Nitrogen Species, and Redox Dependent Signaling in the Cardiovascular System Circulation Research, Vol. 119, No. 5. As non-limiting examples, neutrophils produce myeloperoxidase which converts H2O2 and NaCl into the ROS hypochlorous acid and hypochlorite (HOCl, −OCl)—much more potent free radicals which are antibacterial and an important part of the host defense mechanism but can also damage cell membranes, DNA and proteins (Klebanoff, S. J. (2005). Myeloperoxidase: Friend and Foe. J. Leucocyte Biology. 77: 598-625). Macrophages produce inducible nitric oxide synthase 2 (iNOS) which produces large amounts of nitric oxide (NO). H2O2 and NO combine to form the very powerful RNS peroxynitrite (ONOO−) which is also an important part of the host defense mechanism but also damages membranes, DNA and proteins (Pacher P, Beckmann J S and Liaudet L. (2007). Nitric Oxide and Peroxynitrite in Health and Disease. Phsiol. Rev., 87(1): 315-424).
Mucosal and glandular cells produce lactoperoxidase which catalyzes the conversion of thiocyanate (SCN) into hypothiocyanite (OSCN) in the presence of H2O2. Hypothiocyanite has potent antibacterial activities and appears non-toxic to human cells (Day B J. (2019). The science of licking your wounds: Function of oxidants in the innate immune system. Biochem Pharmacol., 163: 451-457).
The SERPIN B1 variant polypeptides disclosed herein can retain their neutrophil or pancreatic elastase inhibition activity even in the presence of free radicals. Elastase inhibition activity of the variant polypeptides can be tested using methods well known in the art. For example, elastases can be incubated with the SERPIN B1 variant polypeptide, followed by adding an elastase substrate. The elastase cleaves the substrate to produce a colorimetric or fluorescent signal, which can be detected using a suitable device. One exemplary substrate is Succ-AAPV-pNA. Similar assays can be performed with a SERPIN B1 variant polypeptide that has been treated with a free radical (ROS or RNS) or an enzyme or other agent that produces free radicals, e.g., peroxynitrite, hypochlorous acid, or myeloperoxidase, to assess its elastase inhibition activity. The elastase inhibition activity of SERPIN B1 variant polypeptides after being exposed to free radicals is substantially the same as that of the SERPIN B1 variant polypeptides before the exposure. One illustrative example of the elastase inhibition assay is described in Example 1.
The variant polypeptide(s) has an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, %, at least 98%, at least 99% identical to SEQ ID NO: 1 over the full length sequence of SEQ ID NO: 1, and the variant polypeptide is also able to inhibit the protease activity of an elastase (e.g., human neutrophil elastase or a pancreatic elastase). In some embodiments, the variant polypeptide comprises a single amino acid substitution that is selected from the group consisting of C344A, C344V, and C344G and as relative to the native human SERPIN B1 (SEQ ID NO: 1). In some embodiments, the variant polypeptide comprises the sequence of SEQ ID NO: 2, SEQ ID NO:3, or SEQ ID NO: 4.
Sequence identity or similarity disclosed herein may be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman J. of Mol. Biol. Vol. 147, Issue 1:195-197 (1981); the sequence identity alignment algorithm of Needieman & Wunsch; the search for similarity method of Pearson & Lipman; the computerized implementations of these algorithms (GAP, BESTFIT, FASTA, BLAST, Clustal Omega, and TFASTA in the Wisconsin Genetics Software Package, Genetics computer Group, 575 Science Drive, Madison, Wis.); or the Best Fit sequence program described by Devereux et al. Nucleic Acids Res. 12:387-95 (1984), preferably using the default settings. In one embodiment, computerized implementations of the BLAST 2.0 algorithm described in Altschul et al., 1990, J. Mol. Biol. 215:403-410 (using default parameters) may be used to determine the sequence identity.
Sequence identity can also be determined by inspection of the sequences. For example, the sequence identity between sequence A and sequence B, aligned using the software above or manually, can be determined by dividing the sum of the residue matches between sequence A and sequence B by the result of the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, times one hundred.
SERPIN B1 variant polypeptides can be generated by modifying the native polypeptide (SEQ ID NO: 1) according to methods well-known to the skilled in the art. Such methods include, but are not limited to, mutagenesis by PCR, which uses primers designed to contain desired changes; nested primers to mutate a target region; and inverse PCR, which amplifies a region of unknown sequence using primers orientated in the reverse direction. Many other mutation and evolution methods are also available and expected to be within the skill of a person of ordinary skill in the relevant art.
The polynucleotides encoding the SERPIN B1 variant polypeptides described herein may also be chemically synthesized in accordance with the desired sequence by a known synthesis process. These sequences can be cloned into an expression vector using well-established cloning procedures, as further described below.
Chemical or enzymatic alterations of expressed polynucleotides and polypeptides can be performed. For example, sequences can be modified by the addition of lipids, sugars, peptides, organic or inorganic compounds, by the inclusion of modified nucleotides or amino acids, or the like using standard methods. Accordingly, the present invention provides for modification of any of the SERPIN B1 variant polynucleotides or polypeptides by mutation, chemical or enzymatic modification, or other available methods, as well as for the products produced by practicing such methods, e.g., using the sequences herein as a starting substrate for the various modification approaches.
C. Pharmaceutical Compositions Comprising SERPIN B1 or SERPIN B1 Variants
This disclosure also provides pharmaceutical compositions comprising a native SERPIN B1 or a SERPIN B1 variant polypeptide disclosed herein, and one or more pharmaceutically acceptable carriers. The SERPIN variant polypeptide possesses neutrophil elastase inhibition activity. In some embodiments, the neutrophil elastase inhibition activity is resistant to oxidation by free radicals. In some embodiments, the pharmaceutical composition comprises a native SERPIN B1, or a SERPIN B1 variant polypeptide, which has an amino acid sequence that is at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, %, at least 98%, at least 99% identical to SEQ ID NO: 1. In some embodiments, the variant polypeptide comprises a single amino acid substitution that is selected from the group consisting of C344G, C344A, and C344V as compared to the native human SERPIN B1 (SEQ ID NO: 1). In some embodiments, the variant polypeptide comprises the sequence of SEQ ID NO: 2, SEQ ID NO:3, or SEQ ID NO: 4.
In some embodiments, the pharmaceutically acceptable carrier is a reducing agent (e.g., N-acetylcysteine (NAC)), which is capable of preventing C344 of SERPIN B1 polypeptide from being oxidized by the free radicals. In some embodiments, the pharmaceutical composition comprises the native SERPIN B1, which has the sequence of SEQ ID NO: 1 and the reducing agent. In some embodiments, the pharmaceutical composition comprises a variant SERPIN B1, which has an amino acid sequence that is at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, %, at least 98%, at least 99% identical to SEQ ID NO: 1 over the full length sequence of SEQ ID NO: 1, and the variant polypeptide is also able to inhibit the protease activity of a neutrophil elastase (e.g., a human neutrophil elastase) or a pancreatic elastase (e.g. a human pancreatic elastase).
Other pharmaceutically acceptable carriers, excipients, or stabilizers may also be used at suitable dosages and concentrations. These pharmaceutically acceptable carriers, excipients, or stabilizers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as olyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Exemplary formulations are described in WO98/56418, expressly incorporated herein by reference. Lyophilized formulations adapted for subcutaneous administration are described in WO97/04801. Such lyophilized formulations may be reconstituted with a suitable diluent to a high protein concentration and the reconstituted formulation may be administered subcutaneously to the individual to be treated herein. Lipofectins or liposomes can be used to deliver the SERPIN B1 polypeptide or a variant thereof to a patient in need thereof.
The amount of the reducing agent used in the pharmaceutical composition may vary, and the amount should be sufficient to prevent oxidation of C344 of the wild type SERPIN B1 polypeptide or SERPIN B1 variant polypeptides.
D. Treating Diseases with Oxidation-Resistant SERPINS
The SERPIN B1 variant polyptides or pharmaceutical composition comprising thereof can be used to treat a patient having a disease or genetic condition associated with exposure to high levels of free radicals or increased exposure to the free radicals present in environmental sources (e.g. air pollutants including tobacco smoke, vape device emissions). The SERPIN B1 variant polyptides or pharmaceutical composition comprising thereof can also be used to treat a disease or genetic condition that is associated with increased production of free radicals, by e.g., activated endogenous enzymes (e.g. peroxidases or nitric oxide synthases) resident in innate immune cells (e.g. neutrophils, monocytes, macrophages, eosinophils) or tissues and organs as compared to a normal individual.
Non-limiting examples of these diseases are selected from groups of infectious, autoimmune, respiratory, metabolic, cardiovascular, neurodegenerative or oncology diseases. Infectious diseases include, but are not limited to, pulmonary or systemic diseases such as acute lung injury (ALI), acute respiratory distress (ARDS), pneumonia, bronchiolitis, systemic coagulopathies or hemorrhagic diseases caused by, but not limited to, respiratory syncytial viruses, influenza viruses, coronaviruses, ebola viruses, Pseudomonas aeruginosa and other opportunistic pathogens. Autoimmune diseases include, but are not limited to, type 1 diabetes, rheumatoid arthritis, psoriasis, multiple sclerosis and sterile autoinflammatory diseases (SAID's) that have underlying genetic mutation(s) predisposing patients to recurrent bouts of episodic inflammation. Respiratory diseases include, but are not limited to, allergic asthma, smokers' emphysema, COPD and idiopathic pulmonary fibrosis (IPF). Metabolic diseases include, but are not limited to, type 2 diabetes, insulin resistance, dyslipidemia and cataract formation. Cardiovascular diseases include but are not limited to, atherosclerosis and hypertension. Neurodegenerative diseases include, but are not limited to, Parkinson's and Alzheimer's. Oncology diseases include, but are not limited to, colorectal, pancreatic, prostate, breast, lung and bladder cancers. Examples of diseases that are associated with free radicals are also described in Maddu, Disease Related To Types Of Free Radicals, (2019), DOI: 10.5772/intechopen.82879, the entire content of which is herein incorporated by reference.
The pharmaceutical compositions of the wild type SERPIN B1 or SERPIN B1 variant polypeptides may be administered to a subject at a therapeutically effective dose to treat a disease or a genetic condition as described above. The pharmaceutical compositions can be administered by, e.g., inhalation, intra-tracheally, topically or by injection subcutaneously, intravenously, or intraperitoneally.
Generally administered dosage will be one that is effective to achieve the desired therapeutic effect. One of ordinary skill in the art understands that the dose administered will vary depending on a number of factors, including, but not limited to, the subject's body weight, age, individual condition, surface area or volume of the area to be treated, and/or on the form of administration. The size of the dose also will be determined by the existence, nature, and extent of any adverse effects that accompany the administration of a particular compound in a particular subject. Preferably, the smallest dose and concentration required to produce the desired result should be used. Dosage should be appropriately adjusted for children, the elderly, debilitated patients, and patients with cardiac and/or liver disease. Further guidance can be obtained from studies known in the art using experimental animal models for evaluating dosage. In some embodiments, the administered dosage is one that delivers an amount of the native SERPIN B1 (SEQ ID NO: 1) or SERPIN B1 variant polypeptide that range from 0.01-1000 mg/kg, e.g., 0.1-500 mg/kg, 0.5-100 mg/kg, 1.0-50 mg/kg, or from 1.0-25 mg/kg. In some embodiments, the native SERPIN B1 or the SERPIN B1 variant polypeptide as disclosed herein is administered in combination with (e.g., simultaneously or sequentially) a reducting agent disclosed herein. In some embodiments, the reducing agent (e.g., NAC) may be delivered in an amount of 0.01-100 mg per kilogram of the patient's mass.
Optimal dosing schedules can be calculated from measurements of agent accumulation in the body of a subject. In general, dosage may be given once or more daily, weekly, or monthly. Persons of ordinary skill in the art can easily determine optimum dosages, dosing methodologies, and repetition rates. In some embodiments, the compositions of the invention are administered one or more times a day, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times a day. In some embodiments, the compositions of the invention are administered for about 1 to about 31 days, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. In some embodiments, the compositions of the invention are administered for at least 1 day. In other embodiments, the compositions of the invention are administered for one or more weeks, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more weeks. In yet other embodiments, the compositions are administered for one or more months, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months.
To achieve the desired therapeutic effect, the compositions of the invention may be administered for multiple days at the therapeutically effective daily dose. Thus, therapeutically effective administration of the compositions of the invention to treat a pertinent condition or disease described herein in a subject requires periodic (e.g., daily or twice daily) administration that continues for a period ranging from three days to two weeks or longer. While consecutive daily doses are a preferred route to achieve a therapeutically effective dose, a therapeutically beneficial effect can be achieved even if the agents are not administered daily, so long as the administration is repeated frequently enough to maintain a therapeutically effective concentration of the agents in the subject. For example, one can administer the agents every day, every other day, or, if higher dose ranges are employed and tolerated by the subject, twice a week.
A dose can be formulated in animal models to achieve a concentration range that includes the IC50 (the concentration of the agent that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in stool or an enteric tissue sample can be measured, for example, by high performance liquid chromatography (HPLC). In general, the dose equivalent of the active ingredient of the composition of the invention is from about 1 ng/kg to about 1000 mg/kg, e.g., about 1 mg/kg to about 100 mg/kg for a typical subject.
The dosage of a composition of the present invention can be monitored and adjusted throughout treatment, depending on severity of symptoms, frequency of recurrence, and/or the physiological response to the therapeutic regimen. Those of skill in the art commonly engage in such adjustments in therapeutic regimens.
E. Production of the Native SERPIN B1 and its Variants
Optionally, the present disclosure provides coding sequence of the native SERPIN B1 or SERPIN B1 variant polypeptides have been engineered to match the codon usage pattern of the host (e.g., yeast) to maximize expression efficiency. Methods for codon optimization are readily available, for example, optimizer, accessible free of charge at genomes.urv.es/OPTIMIZER, OPTIMUMGENE™ algorithm from GenScript (Piscataway, N.J.), and GENEGPS® Expression Optimization Technology from DNA 2.0 (Newark, Calif.). In one embodiment, the coding sequence is codon-optimized for expression in S. cerevisiae. In some embodiments, the coding sequence is not codon-optimized.
The coding sequences of the SERPIN or its variants can be cloned into an expression vector, such as a plasmid, a cosmid, a phage, a virus (e.g., a plant virus), a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), or the like, into which a nucleic acid sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. In some embodiments, the expression vector is a Yeast episomal expression plasmid (YEp) containing a selectable marker.
In some embodiments, the promoter is a yeast promoter, e.g., the yeast ADH2 promoter. In other embodiments, the vector is an engineered yeast 2 micron plasmid.
Expression vectors comprising the coding sequences disclosed above can be transformed into a variety of host species or strains. In one embodiment, the host species is S. cerevisiae. In another embodiment, the S. cerevisiae is a strain that has been genetically modified to be protease-deficient.
Also provided herein is a method and composition of a fusion protein comprising the native SERPIN B1 or SERPIN B1 variant polypeptide and a second polypeptide. In some embodiments, the second polypeptide increases the half life of the fusion protein. For example, the second polypeptide may comprise or consist of a Fc portion of IgG, a single chain variable fragment (scFv), or an antibody.
In some embodiments, the native SERPIN B1 or SERPIN B1 variant polypeptide is modified post translationally, e.g., by pegylation.
Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a method is disclosed and discussed and a number of modifications that can be made to a number of molecules including the method are discussed, each and every combination and permutation of the method, and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed.
The following examples are for illustrative purposes only and should not be interpreted as limitations of the claimed invention. There are a variety of alternative techniques and procedures available to those of skill in the art which would similarly permit one to successfully perform the intended invention.
Materials—Purified human neutrophil elastase (HNE) and cathepsin G (CatG) were purchased from Lee Biosolutions (St. Louis, Mo.). Porcine pancreatic elastase (PPE), TLCK-treated bovine a chymotrypsin (BC), N-chlorosuccinimide (NCS), the rabbit polyclonal anti-Serpin B1 antibody (PN #SAB1101121) and a goat anti-rabbit HRP conjugate were purchased from Sigma-Aldrich (St. Louis, Mo.). The elastase substrate N-Succ-AAPV-pNA and BC substrate Succ-AAPF-pNA were purchased from Bachem Americas (Torrance, Calif.). 2-Mercaptoethanol (2-ME) was purchased from MP Biomedical (Santa Ana, Calif.). Sodium thiosulphate pentahydrate was purchased from VWR International (Visalia, Calif.). Chromatography Resins QXL fast-flow, Sephacryl S100 HR and Hitrap Q HP were purchased from GE Healthcare Life Sciences (Pittsburgh, Pa.). Hydroxyapatite (Macro-Prep Ceramic, Type 1, 40 um) was purchased from Biorad Laboratories (Hercules, Calif.). The Zorbax 300SB-C3 HPLC column was purchased from Agilent Technologies (Santa Clara, Calif.).
Expression of Recombinant Human sB1's in Yeast—Recombinant human serpin B1 (rhsB1) was expressed in and purified from the yeast S. cerevisiae. The human mRNA sequence encoding wild type human serpin B1 under the control of the yeast ADH2 promoter was synthesized and subcloned into the E. coli vector pUC57 at Genscript USA (New Jersey). The vector (2 ug) was digested with endonucleases and the appropriately sized DNA fragment recovered using a Zymoclean gel DNA recovery kit (Zymo Research, San Diego). This fragment was ligated into a yeast 2 um plasmid (pSB100) containing a ura- selectable marker and transformed into S. cerevisiae. Several colonies that grew on Ura-/8% glucose plates were screened for rhB1 expression by growing overnight at 30° C. in an inoculum of synthetic defined (SD) media (Sunrise Sciences, San Diego) lacking uracil but containing 8% glucose then transferring into yeast extract/peptone/2% glucose (YEPD) and growing for an additional 72 hours. Samples were taken at 24, 48 and 72 hours and analyzed for growth (OD600), protein expression (SDS-PAGE) and rhB1 activity (inhibition of porcine pancreatic elastase). Several clones displaying good expression and activity of rhsB1 were selected and glycerol stocks prepared and frozen at −80° C. The Cys-344→serine mutation was introduced into the human serpin B1 coding sequence in pUC57 by site directed mutagenesis at Genscript USA (New Jersey) substituting C for G1031. This variant sequence was excised, subcloned, and transformed into yeast as described for the wild type rhsB1 protein. The coding sequences of the expression constructs were verified by DNA sequencing.
Purification of Recombinant Human sB1—rhsB1 was purified from yeast paste (50-100 g) using a combination of column chromatography and ammonium sulfate precipitation. As we were interested in the reactivity of the two cysteine residues in sB1 (Cys-214, Cys-344) and how they would behave once liberated from the highly reductive intracellular yeast environment, reducing agents were excluded from all purification steps. In brief, cells were subjected to glass bead lysis in a 10 mM tris buffer, pH 8.0 containing 1 mM EDTA (TE) using a bead beater (BioSpec Products, OK). Lysates were clarified by centrifugation at 20,000×g and the pH adjusted to 8.0 before loading directly onto an anion exchange column containing QXL fast flow resin equilibrated in TE buffer. Bound proteins were eluted with a 5CV gradient to 1M NaCl in the equilibration buffer. Fractions containing active rhsB1 were located using SDS-PAGE and a PPE inhibition assay (see: enzyme inhibition assays section), pooled and further purified and concentrated by consecutive 45% and 65% ammonium sulfate (AS) precipitation steps. For each of these steps solid AS was added to the rhsB1 containing pool, mixed at room temperature for 30 minutes then centrifuged for 20 minutes at 20,000×g. The 65% AS pellet was re-dissolved in a minimal volume of 10× TE buffer, pH 8.0 and loaded onto a size exclusion column containing Sephacryl 5100 HR resin equilibrated in TE, 100 mM NaCl, pH 7.4. Proteins were eluted at 5 mL/min and the peak containing monomeric rhB1 located by SDS-PAGE analysis. Peak fractions were pooled, dialyzed overnight into 10 mM NaCl, 5 mM sodium phosphate, pH 6.9 and loaded onto a column of ceramic hydroxyapatite (CHA) equilibrated in the dialysis buffer at 5 mL/min. The column was washed with equilibration buffer and bound proteins eluted with a 20 column volume gradient to 10 mM NaCl, 0.3M sodium phosphate, pH 6.9. Peaks eluting from the column were tested for rhsB1 content by SDS-PAGE and PPE inhibition. Fractions containing rhsB1 were pooled and concentrated on Vivaspin 20 ultrafiltration spin columns (Sartorius, Germany). Protein concentration was determined using the published extinction coefficient of 1.16 for a 1 mg/mL solution of rhsB1 and aliquots frozen at −80° C. (12).
Sensitivity of rhsB1 in Yeast Lysates to oxidation with N-chlorosuccinimide (NCS)—100 mg samples of yeast paste expressing rhsB1 were lysed by mechanical disruption. To a microfuge tube containing yeast paste, 0.75 mL of glass beads (0.5 mm) and 0.75 mL of TE (pH 8.0) were added and the tube mixed on a mini-vortexer (VWR scientific) for 3 pulses of 1 min each with 1 min rests on ice between pulses. Lysates containing soluble rhsB1 were clarified by centrifugation at 20,000×g for 10 min. Aliquots of clarified lysate (90 uL) were made to various concentrations of NCS (0-1 mM) by adding 10 uL of a 10× NCS concentrate (in TE, pH 8.0) to each aliquot and incubating at 30° C. for either 1, 5 or 30 minutes. Reactions were stopped by adding an excess of 1.0M sodium thiosulphate. Samples were taken and assayed for inhibition of porcine pancreatic elastase (PPE) and bovine chymotrypsin (BC) in PBS and analyzed by SDS-PAGE and western blotting for rhsB1 molecular form.
Porcine Pancreatic Elastase (PPE) assay—Inhibition of PPE was used both as a tool to detect rhsB1 containing fractions during purification and as an assay to assess the relative PPE inhibitory activity of PTM forms of rhsB1 present in oxidized yeast lysates and highly purified protein preparations. To monitor the purification steps, varying amounts of selected chromatographic peak fractions were incubated with PPE (100 or 200 nM) for up to 5 minutes in a final volume of 195 uL of PBS, pH 7.4 in a microtiter plate with or without 2-ME. The elastase substrate Succ-AAPV-pNA was then added to a final concentration of 1 mM and the release of free para-nitroanilide (pNA) monitored for several minutes at 405 nm on a plate reader (SPECTRAmax 340PC, Molecular Devices). Fractions containing rhsB1 were noted as those that fully inhibited PPE and were pooled and further processed as described in the purification section. When used as an assay to assess the relative inhibitory activity of rhsB1 proteins present in oxidized yeast lysates and highly purified preparations, a fixed concentration of PPE was used (100 nm). PPE was incubated with varying volumes of each sample in a fixed volume of PBS, pH 7.4 (195 uL) in a microtiter plate with or without 2-ME for varying time periods. Residual PPE activity was measured as described above. Similar assays can be performed assess the inhibitory activity of SERPIN B1 variants on a human pancreatic elastase.
Human Neutrophil Elastase (HNE) Assay—Human neutrophil elastase (HNE; 170 nM) was incubated with varying volumes of oxidized or non-oxidized yeast lysates and varying amounts of purified rhsB1 protein preparations in a fixed volume (195 uL) of PBS, pH 7.4 in a microtiter plate with or without 2-ME for varying time periods. Residual HNE activity was determined by adding the elastase substrate Succ-AAPV-pNA and monitoring as described above.
Bovine Chymotrypsin Assay—Bovine chymotrypsin (BC; 100 nM) was incubated with varying volumes of oxidized or non-oxidized yeast lysates and varying amounts of purified rhsB1 protein preparations in a fixed volume (195 uL) of PBS, pH 7.4 in a microtiter plate with or without 2-ME for varying time periods. Residual BC activity was determined by adding the BC substrate Succ-AAPF-pNA and monitoring as described above.
Human neutrophil Cathepsin G (CatG) Assay—Human neutrophil cathepsin G (CatG; 100 nM) was incubated with varying amounts of purified rhsB1 protein preparations in a fixed volume (195 uL) of PBS, pH 7.4 in a microtiter plate with or without 2-ME for varying time periods. Residual CatG activity was determined by adding the substrate Succ-AAPF-pNA and monitoring as described above for BC
Interaction of PPE, HNE, BC and CatG with rhsB1 proteins—Enzyme:rhsB1 complexes were visualized by SDS-PAGE and staining with coomassie blue G250. Typically, a fixed amount of enzyme was incubated with varying amounts of purified rhsB1 protein or a fixed amount of purified rhsB1 protein was incubated with varying amounts of enzyme for varying time periods sufficient to achieve either maximal complex formation or maximal conversion of rhsB1 proteins to lower molecular weight forms (based on the published association rate constants (4) and the data obtained from the enzyme assays described above). Residual enzyme was inhibited by the addition of a synthetic low molecular weight protease inhibitor cocktail (Sigma) and samples analyzed by SDS-PAGE.
Protein Sequence analysis of cleaved rhsB1M* and rhsB1D—Purified rhsB1M* and rhsB1D cleaved by PPE were further purified by ion exchange chromatography on a Hitrap Q HP column (5 mL) to remove PPE and residual low molecular weight synthetic protease inhibitors. Samples of each were sequenced through 7 cycles by Edman degradation at the UC Davis Molecular Structure Analysis Facility (Davis, Calif.).
5 uL of yeast lysate containing rhsB1 were incubated and oxidized with varying concentrations of NCS.
rhsB1 in yeast lysates was sensitive to oxidation with NCS (as determined by the loss of EIA) which induced a molecular weight shift similar to that seen in rhsB1M*. Aliquots (5 uL) of untreated lysates were able to completely inhibit PPE (100 nM) activity but this EIA was rapidly and specifically lost in a dose dependent manner upon oxidation with NCS (
In the absence of 2-ME neither rhsB1M* nor rhsB1D were able to inhibit PPE. Instead, both were readily cleaved to lower molecular weight species.
In the presence of 25 mM 2-ME, rhsB1M* was still unable to efficiently inhibit PPE and was also degraded to the lower molecular species as described above (data not shown). In contrast, rhsB1D was reduced by 25 mM 2-ME to an active monomeric rhsB1 able to form SDS-stable complexes with PPE in a dose dependent manner (
Direct protein sequence analysis of rhsB1M* cleaved by PPE yielded 3 separate but overlapping sequences derived from the RSL of sB1 (Table 1,
No protein sequence data was obtained from the N-terminal amino acids of rhsB1 most likely due to acetylation of the N-terminal methionine's α-amino group by N-acetyltransferase B (NatB) interfering with the Edman degradation process chemistry. This has been observed for other N-acetylated clade B serpins and proteins expressed intracellularly (29).
To accurately evaluate the inhibitory activity of fully reduced rhsB1D or rhsB1M* on human neutrophil elastase (HNE) we titrated a fixed amount of each (230 or 460 nM) with differing concentrations of 2-ME and then reacted them with a fixed amount of HNE (170 nM). Appropriate controls evaluating the effect of 2-ME alone on HNE activity were included.
In contrast, titration of 230 nM rhsB1M* with 2-ME only had a small effect on EIA at any of the concentrations tested (
The interaction of rhsB1D and rhsB1M* with HNE provided results very similar to those described above for PPE. Neither were able to significantly inhibit HNE in the absence of a reducing agent and both displayed degradation patterns similar to those seen in
In contrast, the SOI of reduced rhsB1M* with HNE is ˜13 (data not shown) with the majority of rhsB1M* being catalytically cleaved within the RSL and a small amount of intermediate ˜57 kDa complex visible on SDS-PAGE (
In contrast to the elastases, BC was inhibited by both rhsB1D and rhsB1M* in the presence or absence of 2-ME although their SOI's were altered. Our kinetic data indicated that in the presence of 2-ME, rhsB1D had an SOI approaching 0.5 (
The SOI for rhsB1M* with BC was ˜3 in the absence or presence of 2-ME with a trend to a slightly lower SOI in the presence of 2-ME (
As CatG is closely related to chymotrypsin we anticipated that its' interaction with rhsB1D and rhsB1M* would generate similar data to that described above for BC and rhsB1D and rhsB1M*. The results did show a similar interaction kinetically, and by SDS-PAGE analysis of the reaction products. The reduced dimer appeared twice as effective at inhibiting CatG when compared to the unreduced dimer although the SOI for each was approximately double that described for the interaction of rhB1D with BC. This likely reflects either a higher specific activity of the CatG preparation, a higher turnover of rhB1D by CatG during complex formation or, a combination of the two. In support of the second hypothesis, it would appear from the comparative SDS-PAGE profiles in
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, sequence accession numbers, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
This experiment was conducted to analyze the effect of physiologically relevant ROS and RNS on the elastase inhibition activity of sB1. Yeast expressing wild-type serpin B1 (rhsB1 WT (C344) were lysed in PBS, 1 mM EDTA, pH 7.5 and the soluble lysate containing wild-type (C344) serpin B1 separated from insoluble cell debri by centrifugation. Enzyme (PPE) was titrated with the lysate until no PPE activity was observed. The conditions in this experiment were similar to those described in Example 1 and the rhsB1 was present as an unoxidized free monomer rhsB1 C344-SH stabilized by endogenous anti-oxidants present in the yeast lysate. Separate samples of the lysate were treated with varying concentrations of each ROS/RNS for varying times and the reaction stopped with an excess of sodium thiosulphate. Oxidized lysate samples were then reacted with PPE to re-assess inhibitory activity after adjusting for treatment related volume changes using the method described in the legend for
Next we performed an experiment to test the ability of oxidized wild-type sB1 to inhibit other proteases. Yeast lysate containing wild-type serpin B1 (C344) that had been treated and inactivated with peroxynitrite was used to assess its ability to inhibit bovine α-chymotrypsin (
We then purified peroxynitrite inactivated wild-type serpin B1 and tested its ability to inhibit neutrophil proteinase 3 (PR3;
Serpin B1 reactive site variants were constructed by changing the DNA coding sequence for the wild-type amino acid (C344) by site-directed mutagenesis. Sequences were verified by DNA sequencing, cloned into a yeast expression vector and transformed into the yeast S. cerevisiae. Variant proteins were expressed and purified as previously described.
Human Alpha 1-Antitrypsin (AAT) was included as a reference control. The molecular weight of AAT is approx. 54,000 daltons compared to approx. 42,500 daltons for serpin B1.
Enzyme assays: A concentration of porcine pancreatic elastase (PPE) was used that yielded a Vmax of ˜400 mAU/min at 405 nm with the chromogenic substrate Succ-AAA-pNA in a final volume of 200 uL in a microtiter plate. This was typically 160 nM. The order of addition of reagents to a microtiter plate well was as follows: 1. Enzyme (8-10 uL of an 0.1 mg/mL working stock of PPE); 2. Buffer (PBS, pH7.4, +40 mM 2-mercaptoethanol); 3. Inhibitor. The solution was mixed, incubated for 5 min at 25° C., then 5 uL of chromogenic substrate was added to a final concentration of 1 mM and enzyme activity recorded for 2 min on a SpectraMax 340 PC microtiter plate reader (Molecular Devices). Each assay was performed in triplicate on 2 occasions. Results showed that all four variants exhibit elastase inhibition activity but they do so with very different efficiencies (
In this experiment a fixed amount of wild-type rhsB1 (C344) or the C344S variant (S344) were incubated with varying amounts of PPE at the molar ratios indicated. The reaction volume was fixed at 20 uL. PPE was added first followed by buffer and inhibitor. Samples were incubated for 5 min at 25° C. then a synthetic protease inhibitor mix (Sigma cat #P8215) added to quench active enzyme, SDS-PAGE loading buffer added and samples heated for 5 min. Reaction products were resolved by 4-20% SDS-PAGE and visualized by staining with coomassie blue R250 (
The C344A rhsB1 Variant is Resistant to Oxidation
In this experiment yeast lysates containing either wild-type serpin B1 (C344) or the C344A variant were treated with the oxidant peroxynitrite as described previously. Samples were then tested for their ability to inhibit elastase and chymotrypsin as described in
In this experiment yeast expressing either serpin B1 wild-type protein (rhsB1 WT) or the C344A variant (rhsB1 A344) were lysed in PBS, pH 7.5. Enzyme (PPE) was titrated with lysate(s) until no PPE activity was observed. Lysate(s) were then incubated with 1 or 5 U of purified human neutrophil myeloperoxidase (MPO)+25 mM sodium chloride and 80 uM H2O2 for varying times and the reaction stopped with an excess of sodium thiosulphate. Oxidized lysate samples were reacted with PPE to re-assess inhibitory activity as previously described. Purified human AAT (hAAT) was used as a control as it has previously been shown to be inactivated by MPO generated free radicals. The results (shown in
2. Remold-O'Donnell, E., Nixon, J. C., and Rose R. M. (1989) Elastase inhibitor: Characterization of the human elastase inhibitor molecule associated with monocytes, macrophages and neutrophils. J Exp. Med. 169, 1071-1086
All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Although the present invention is described primarily with reference to specific embodiments, it is also envisioned that other embodiments will become apparent to those skilled in the art upon reading the present disclosure, and it is intended that such embodiments be contained within the present inventive methods.
The application claims the benefit of, and priority to, U.S. Provisional Application No. 62/881,858, filed on Aug. 1, 2019. The entire content of said provisional application is herein incorporated by reference for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/044604 | 7/31/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62881858 | Aug 2019 | US |
