Patent Application
20060269538
The present invention provides variants of serine proteases with altered sensitivity to one or more activity-modulating substances. A method for the generation of such proteases is disclosed, comprising the provision of a protease library encoding polynucleotide sequences, expression of the enzymes, screening of the library in the presence of one or several activity-modulating substances, selection of variants with altered sensitivity to one or several activity-modulating substances and isolation of those polynucleotide sequences that encode for the selected variants.
Today many severe medical conditions remain untreatable and require innovative new approaches complementing the traditional medicinal chemistry development of drugs. One alternative emerges from the recent successful introduction of biological therapeutics for the treatment of a number of diseases. Examples for biological therapeutics (“biologics”) comprise peptides, proteins, polynucleic acids, lipids or combinations thereof. Traditionally, biologics replaced the bodies own missing or inactive proteins. The potential of biologics has been dramatically broadened by the use of molecules with functions that are not present in the bodies own repertoire, e.g. antibodies directed against a number of targets which are inactivated by binding. In addition, enzymes with different catalytic functions have been developed that increase the rate of a desired reaction with a positive effect on the condition of the patient.
However, the activity of enzymes is highly regulated in the human or animal body at different levels. For example, the expression of an enzyme may be stimulated by activation of transcription factors, or an enzyme may be activated by a reversible posttranslational modification such as phosphorylation. In signal transduction kinase cascades are known in which upstream kinases phosphorylate and thereby activate downstream kinases. The biological effect is downregulated by the action of phosphatases which remove the phosphate residue and render the kinase inactive. The situation is different for example in proteolytic cascades such as known from the coagulation or complement cascade. The proteases are expressed as inactive proenzymes and are activated by proteolytic cleavage. In this case the downregulation of the protease activity is accomplished by the interaction with inhibitors which are present in blood or other body fluids and tissue at high concentrations. The inactivated proteases are degraded and cleared from the bloodstream.
With respect to applications as biological therapeutics proteases represent a particularly promising example as they can specifically activate or inactivate proteins that are involved in a disease or disease symptoms.
While antibodies bind targets in a fixed stoichiometry, a protease can activate or inactivate hundreds or thousands of target proteins. Therefore lower doses can be given with the potential of less side effects and lower manufacturing costs. Since nature does not provide proteases which cleave arbitrary targets of interest with sufficient specificity, ways of generating such specific proteases by molecular techniques have been devised. Specificity is an essential element of enzyme function. A cell consists of thousands of different, highly reactive catalysts. Yet the cell is able to maintain a coordinated metabolism and a highly organized three-dimensional structure. This is due in part to the specificity of enzymes, i.e. the selective conversion of their respective substrates. Specificity is a qualitative and a quantitative property. In nature, the specificity of an organism's enzymes has been evolved to the particular needs of the organism. Arbitrary specificities with high value for therapeutic, research, diagnostic, nutritional or industrial applications are unlikely to be found in any organism's enzymatic repertoire due to the large space of possible specificities. Therefore, defined specificities have to be generated de novo.
The application of therapeutic proteases in the treatment of diseases requires their activity in the presence of activity-modulating substances that are present in the application matrix where enzymatic activity is required, e.g. blood serum, extracellular fluid, cerebrospinal fluid, the intracellular environment, or any other environment in the body where activity is required. The serum in particular contains a large variety of protease inhibitors present in high concentrations, most notably serpins (serine protease inhibitors such as alpha1-antitrypsin, antithrombin, antiplasmin, and others) and macroglobulins (such as alpha2-macroglobulin, and others). While serpins inhibit predominantly serine and cysteine proteases, macroglobulins inhibit also other proteases such as metallo proteases.
There are proteases with lower sensitivity to protease inhibitors then others. A comparatively low sensitivity towards serum inhibitors when comparing it with other human proteases such as trypsin or chymotrypsin has been described for mesotrypsin, a human trypsin variant expressed in the brain and pancreas (Rinderknecht H. et al. Mesotrypsin: A new inhibitor-resistant protease from a zymogen in human pancreatic tissue and fluid. Gastroenterology (1984) 86:681-92). Another example for a protease with comparatively low sensitivity is granzyme B, a serine protease in granules of cytotoxic T-lymphocytes. Kurschus et al. report a 40%-50% residual activity of granzyme B in a solution that corresponds to 80% human serum (Kurschus et al. Killing of target cells by redirected granzyme B in the absence of perforin FEBS Letters (2004) 562:87-92). However, more recent studies have shown that the activity of these proteases in human application matrices containing natural levels of protease inhibitors is not high enough to obtain sufficient activity. And, their specificity is likely to be different from what the application requires.
Besides the therapeutic use, proteases can be used in industrial, cosmetic, diagnostic or synthetic applications. To qualify as an effective protease, in particular for therapeutic purposes, proteases should have a low sensitivity, preferably they should be essentially insensitive, to activity-modulating substances present in the targeted application matrices. Therapeutic protease should be insensitive towards different activity-modulating substances to a degree that provides an activity level sufficient to effect its indicated function and at the same time must have sufficient specificity to avoid side effects.
By a specific screening process novel serine proteases with altered sensitivity to one or more activity-modulating substances were identified. The present invention thus provides
(1) a protease with reduced sensitivity towards activity-modulating substances being derived from a serine protease of the structural class S1 and having one or more mutations at positions selected from the group of positions that correspond structurally or by amino acid sequence homology to the regions or positions 18-28, 34-41, 46-68, 78, 90-102, 110-120, 123-137, 162-186, 195 or 214 in wild-type human cationic trypsin with the amino acid sequence shown in SEQ ID NO:5, or a modified form thereof;
(2) a DNA encoding the protease as defined in (1) above;
(3) a vector comprising the DNA as defined in (2) above;
(4) a cell transformed/transfected with the vector as defined in (3) above and/or containing the DNA as defined in (2) above;
(5) a method for preparing the protease as defined in (1) above, which method comprises cuturing the cell as defined in (4) above 0 and isolating the protease from the culture broth and/or the cell culture;
(6) a pharmaceutical, diagnostic or cosmetic composition comprising the protease as defined in (1) above;
(7) a method for treating a patient in the need of a protease therapy, said method comprising administering the patient a suitable amount of the protease as defined in (1) above; and
(8) a method for generating a protease, preferably a protease as defined in (1) above, having reduced sensitivity towards activity-modulating substances present within an application matrix, comprising
In the framework of this invention the following terms and definitions are used.
The term “polynucleotide” corresponds to any genetic material of any length and any sequence, comprising single-stranded and double-stranded DNA and RNA molecules, including regulatory elements, structural genes, groups of genes, plasmids, whole genomes, and fragments thereof.
The term “site” in a polynucleotide or polypeptide refers to a certain position or region in the sequence of the polynucleotide or polypeptide, respectively.
The term “position” in a polynucleotide or polypeptide refers to specific single bases or amino acids in the sequence of the polynucleotide or polypeptide, respectively.
The term “region” in a polynucleotide or polypeptide refers to stretches of several bases or amino acids in the sequence of the polynucleotide or polypeptide, respectively.
The term “polypeptide” comprises proteins such as enzymes, antibodies and the like, medium-length polypeptides such as peptide inhibitors, cytokines and the like, as well as short peptides down to a amino acid sequence length below ten, such as peptidic receptor ligands, peptide hormones, and the like.
The term “protease” means any protein molecule catalyzing the hydrolysis of peptide bonds. It includes naturally-occurring proteolytic enzymes, as well as protease variants. It also comprises any fragment of a proteolytic enzyme, or any molecular complex or fusion protein comprising one of the aforementioned proteins.
The term “protease variants” means any protease molecule obtained by site-directed or random mutagenesis, insertion, deletion, recombination and/or any other protein engineering method, that leads to proteases that differ in their amino acid sequence from the parent protease.
The “parent protease” can be either an isolated wild-type protease, or one or more protease variants selected from a library of proteases.
The term “protease library” describes at least one protease variant or a mixture of proteases in which every single protease, resp. every protease variant, is encoded by a different polynucleotide sequence.
The term “gene library” indicates a library of polynucleotides that encodes the library of proteases.
The term “isolated” describes any molecule separated from its natural source.
The term “specificity” means the ability of an enzyme to recognize and convert preferentially certain substrates. Specificity can be expressed qualitatively and quantitatively. “Qualitative specificity” refers to the chemical nature of the substrate residues that are recognized by an enzyme. “Quantitative specificity” refers to the number of substrates that are accepted as substrates. Quantitative specificity can be expressed by the term s, which is defined as the negative logarithm of the number of all accepted substrates divided by the number of all possible substrates. Proteases, for example, that accept preferentially a small portion of all possible peptide substrates have a “high specificity”. Proteases that accept almost any peptide substrate have a “low specificity”.
The term “catalytic activity” describes quantitatively the conversion of a given substrate under defined reaction conditions.
The term “activity-modulating substance” describes all substances that, when present in the reaction mixture, physically interact with the protease and alter its catalytic activity compared to the activity in the absence of the substance when all other parameters are kept constant. It therefore comprises all modulators, activators and inhibitors of a protease, and all substances that otherwise alter catalytic activity.
The term “inhibitor” describes all substances that, when present in the reaction mixture, physically interact with a protease and decrease its catalytic activity compared to the activity in the absence of the substance when all other parameters and concentrations are kept constant.
The term “activator” describes all substances that, when present in the reaction mixture, physically interact with a protease and increase its catalytic activity compared to the activity in the absence of the substance when all other parameters and concentrations are kept constant.
The term “application matrix” represents all compositions of molecules, fractions or isolated components that the protease is contacted with at the site where activity is required and during its transfer from the site of first contact with the medium assigned for the specific use and the site where activity of the protease is required. A composition of molecules denotes the entirety of molecules, in particular in their respective combinations and concentrations present at a particular point in space and time. The application matrix comprises both activity-modulating substances, in particular inhibitors or activators, and other activity-modulating substances as well as further components.
The term “compartmentation of samples” describes the coupling of protease genotype and phenotype by use of devices or tools that enable compartmentation of samples. The distribution of genotypes, e.g. into sample carriers is done at a multiplicity per compartment that allows sufficient differentiation of phenotypes.
The term “substrate” or “peptide substrate” means any peptide, oligopeptide, or protein molecule of any amino acid composition, sequence or length, that contains a peptide bond that can be hydrolyzed catalytically by a protease. The peptide bond that is hydrolyzed is referred to as the “cleavage site”.
The term “correspond structurally” refers to amino acid residues or regions of amino acid residues that are located at equivalent positions when performing either a 3-dimensional alignment of structures of human cationic trypsin and structures of other members of the S1 serine protease class or a one-dimensional sequence alignment of human cationic trypsin with the respective proteases. Particular proteins corresponding structurally with the human cationic trypsin of SEQ ID NO:5 are human anionic trypsin und human mesotrypsin shown in SEQ ID NOs:6 and 7, respectively. The respective alignment is shown in
The term “Ki” defines the affinity of an inhibitor “I” to the enzyme “E”. A general kinetic description for a competitive inhibitor is given by the following scheme, whereby “S” indicates the substrate and “p” the product:
Ki is defined as Ki=[E] [I]/[EI]. It represents the dissociation constant of the inhibitor and the enzyme. A large value for Ki corresponds to a weak inhibitor, a small value represents a strong inhibitor.
The term “residual activity” is defined defined as the the ratio of the catalytic activity of the enzyme in the presence of an inhibitor (vi) to the catalytic activity in the absence of the inhibitor (v0), all other parameters being equal. Therefore the residual activity ai is given by ai=vi/v0. and ai*100 is the residual activity in percent. From the above scheme a general equation relating the residual activity to the concentrations of inhibitor and substrate as well as to Km and Ki can be derived:
where Km=[E] [S]/[ES], Ki=[E] [I]/[EI] and vi/v0*100 represents to the residual acitvity in percent.
The term “IC50” is defined as the concentration of activity-modulating substance at which the activity of a protease is reduced to 50% compared to the activity in the absence of the activity-modulating substance, all other parameters and concentrations being equal. In the context of the equation given above this means that [I]=IC50 when vi/v0=½:
which can be transformed in the following way
As set forth above the present invention provides serine protease variants of the structural class S1 with reduced sensitivity towards activity-modulating substances as present in the application matrix of the protease variant and provides a method for the generation of such proteases. The method can be applied to proteases belonging to any known protease class, or sub-class thereof, namely aspartic, cysteine, serine, metallo and threonine proteases. Preferably the method is applied to serine protease of the structural class S1 as disclosed below in Table 2.
Due to the high degree of structural conservation between S1 proteases the substitutions disclosed here for the human cationic trypsin scaffold, may be transferred to other S1 proteases. Namely, substitutions in other scaffolds of the serine protease class S1 at positions that correspond structurally and/or by sequence homology to the positions and/or substitutions disclosed here for human cationic trypsin that to lead to a decreased sensitivity to inhibitors may have an influence on their respective inhibitor-insensitivity of these other scaffolds.
Library Generation
In a preferred embodiment the protease is derived from human trypsin which is sensitive to a variety of inhibitors in the blood, most notably the serpins. Said proteases may have a desired catalytic activity and or substrate specificity but undesired sensitivity to the activity-modulating substances. The invention provides a method to identify and select proteases with a desired change in the sensitivity against said substances.
According to the invention this is achieved by providing a protease library derived from one or more parent proteases with desired catalytic activity, contacting said proteases with at least one activity-modulating substance and selecting one or more protease variants with improved IC50 compared to the parent protease(s).
The first step in selecting proteases with reduced sensitivity towards activity-modulating substances is the generation of libraries of polynucleic acids that encode proteases with different genotypes and/or phenotypes. Different strategies of introducing changes in the coding sequences are applied including but not limited to single or multiple point mutations, exchange of single or multiple nucleotide triplets, insertions or deletions of one or more codons, homologeous or heterologeous recombination between different genes, fusion of additional coding sequences at either end of the encoding sequence or insertion of additional encoding sequences or any combination of these methods. The selection of sites to be mutagenized is based on different strategies as detailed in the following embodiments of the invention. The manipulation of the polynucleic acids to implement these strategies are described in the following embodiments of this first step.
In a first embodiment the generation of libraries is based on the comparison of two or more genes that are different with respect to the sensitivity towards activity-modulating substances. Changes in the gene of interest are then introduced at sites where the amino acid sequences of the two or more proteases differ. The change can result in substitution of one or more amino acids or randomization at these positions or randomization of amino acids one, two or three amino acids upstream and/or downstream from these positions. The same applies to insertions or deletions of one or more amino acids at such positions or any combination of substitution, insertion and deletion.
In a further embodiment the strategy is guided by the analysis of the crystal structure, if available, of the complex between the protease and an activity-modulating substance. The distances between atoms belonging to the protease and those belonging to the activity-modulating substance are analyzed and ranked. In a preferred aspect of this embodiment positions are identified that correspond to amino acids whose atoms have a less than a minimal distance to the closest atom of the activity-modulating substance. Either these positions or amino acids in addition to one, two or three amino acids upstream and/or downstream are randomized, or amino acids are inserted or deleted at these positions or any combination of these changes. The minimal distance of the atoms is less than 10 Å. In a more preferred embodiment the minimal distance is less than 5 Å. If no structure of a complex is available such structure is computer modelled from structures of proteases and/or inhibitors that are related to the proteases and/or inhibitors of interest.
The next embodiment is based on the identification of amino acids which are near the active site and located on the surface of the molecule as preferred sites of mutagenesis. In this embodiment the active site of the protease is identified and a line drawn from the center of mass of the molecule through the center of the active site. A plane perpendicular to this line is approached stepwise from a distant position to the protease towards the open side of the active site. As the plane approaches the protease it will come closer to certain amino acids of the structure. As the plane is approached further it will contact successively more amino acids. The amino acids that are contacted first are the preferred sites for the introduction of mutations. Either these positions or amino acids in addition to one, two or three amino acids upstream and/or downstream are randomized, or amino acids are inserted or deleted at these positions or any combination of these changes.
In another embodiment the sites targeted for the introduction of changes in the gene are random. Such random point mutations are introduced into the gene of interest by means of mutagenic PCR. Depending on the desired mutation spectrum, this can be accomplished either by a method analogous to the protocol of Cadwell and Joyce (Cadwell R C and Joyce G F. Mutagenic PCR PCR Methods and Applications (1994) 3:136-140; Cadwell R C and Joyce G F. Randomization of Genes by PCR Mutagenesis PCR Methods and Applications (1992) 2:28-33), or by the method of Spee et al. (Spee J H et al. Efficient random mutagenesis method with adjustable mutation frequency by use of PCR and dITP Nucleic Acid Research (1993) 3:777-778), or by similar methods or methods derived thereof.
According to a further embodiment, primer extension PCR is utilized to introduce certain changes into a gene basically as described by Ho et al. (Ho S N et al. Site-directed mutagenesis by overlap extension using the polymerase chain reaction Gene (1989) 77:51-59 and Horton R M et al. Engineering hybrid genes without the use of restriction proteases: gene splicing by overlap extension Gene (1989) 77:61-68) or a method derived thereof. The method is applied to mutagenize one or more codons, or to insert one or more codons, or to accomplish complete codon mutagenesis.
In a further embodiment, selective combinatorial randomization (SCR®) is applied for saturating mutagenesis at specific positions within the gene of interest as described in EP 1419248 B1. Using this method, the region to be randomized is determined by a base pair mismatch within a DNA fragment. This can be generated by annealing complementary single strands of different gene variants forming a heteroduplex. The mismatch position is then recognized and selectively randomized.
In the next embodiment, several variants which were generated by a method analogous to one or more of the above embodiments are recombined by recombination chain reaction (RCR®) as described in EP 1230390 B1. Using this method, two largely complementary single strands of different gene variants are annealed. The generated heteroduplex is partially digested by an exonuclease and resynthesized with a polymerase, thus adopting the sequence of one strand into the other one during the extension reaction.
In order to generate enzyme variants with different phenotypes, the libraries of polynucleic acids that encode these different protease variants are translated into proteins by different means.
Therefore, a suitable host cell is transformed with the encoding polynucleic acid and cultivated under appropriate conditions leading to expression and possible secretion of the protease variant. Different organisms may function as hosts including mammalian or non-mammalian cell lines, microbial organisms or viral expression systems. In a preferred embodiment expression is performed in a microbial system such as yeasts, fungi or bacteria. In a preferred embodiment a bacterial host, preferably Echerichia coli or Bacillus subtilis is used. Alternatively, the expression is performed applying a viral expression system and in a preferred embodiment a viral display system is used. In addition, a further embodiment comprises in-vitro translation and transcription systems that allow the generation of active protein from the polynucleic acid in the absence of any living organism.
In another embodiment the coupling between genotype and phenotype is performed by surface display expression methods. Such methods include, for example, phage or viral display, cell surface display and in vitro display. Phage or viral display typically involves fusion of the protease to a viral/phage protein. Cell surface display, i.e. either bacterial or eukaryotic cell display, typically involves fusion of the protease to a peptide or protein that is located at the cell surface. In in-vitro display, the protease is typically made in vitro and linked directly or indirectly to the mRNA encoding the protein (DE 19646372 C1). With phage panning as described by Russel et al. (Russel M, Lowman H B, Clackson T. Introduction to phage biology and phage display, In: Clackson T, Lowman H B, editors. Phage display—a practical approach. Oxford: Oxford University Press; 2004:1-26) the protease is displayed a fusion molecule to a phage surface protein, e.g. as N-terminal part of the gIII surface protein of bacteriophage M13. This can be accomplished by fusing a protease gene library to the C-terminal fragment of the gene gIII and inserting this construct into a phagemid vector. After transformation into an Escherichia coli strain phage particles can be obtained by infection with a helper phage. In a preferred embodiment this procedure is performed with a library of enzyme variants wherein all variants have a defined mutation in the active site rendering the proteases catalytically inactive.
The recovery of the polynucleic acid that encodes the protease with the desired properties requires a strict coupling of the genotype with the protein and its phenotype.
In one embodiment this is performed by separating individual transformants of the host cells or individual viruses into isolated compartments of any type followed by cultivation and expression of the protease variants therein. In a preferred embodiment these compartments are given by the individual wells of a micro titer plate, in a more preferred embodiment this is a high-density micro titer plate of any format.
In another embodiment coupling of genotype and phenotype is obtained by in-vitro transcription and translation of individual polynucleic acids isolated in individual compartments which can be represented by the wells of microtiter plates or droplets of water-in-oil, or water-oil-water emulsions (Tawfik D S and Griffiths A D. Man-made cell-like compartments for molecular evolution Nature Biotechnology (1998) 16:652-656; Bernath K et al. In vitro compartmentalization by double emulsions: sorting and gene enrichment by fluorescence activated cell sorting Analytical Biochemistry (2004) 325:151-157).
Selection of Proteases
In the second step the multitude of expressed proteases are contacted with the at least one activity-modulating substance. Either simultaneously or consecutively the proteases are contacted with at least one substrate.
The consecutive contact is preferred when preselection of a subset of proteases that interact to a lower extent or do not interact with the activity-modulating substance is required. Therefore, the proteases are contacted first with the at least one activity-modulating substance and preincubated. Proteases that interact to a lower extent or do not interact with the activity-modulating substance are selected within a subset. This subset of proteases is subsequently contacted with the at least one substrate to identify those variants that are catalytically active. The contact with the at least one substrate is performed either alone or in combination with the activity-modulating substance. In contrast simultaneous contact of the proteases with the at least one activity-modulating substance and the at least one substrate allows the direct determination of the catalytic activity in the presence of the activity-modulating substance.
The application matrix and therefore the activity-modulating substances depends on the use of the selected proteases. Proteases can be used in industrial, cosmetic, diagnostic or synthetic applications. For these uses the application matrix is given by the composition of any environment relevant for the industrial process, any composition of a cosmetic product or diagnostic reagent, or compositions used in a synthetic application. In one embodiment the proteases are used in the generation of hydrolysates of protein from different plant or animal sources, such as soy, casein and rice. These contain a significant amount of protease inhibitors, e.g. the soy protease inhibitors, Bowman-Birk protease inhibitors (BBI), or soybean trypsin1 inhibitor (SBTI), which reduce the activity of the processing enzyme. Similarly, proteases are used on bakery to enhance the dough properties. The ingredients of the dough, namely flour, contain inhibitors for proteases and other enzymes. The method of the invention provide proteases with reduced inhibitor sensitivity and favourable process performance.
Applications of Proteases
A preferred use of the proteases selected by the method of the present invention is as pharmaceutically active substances that reduce or cure the cause or symptoms of a disease. Depending on the indication for which a pharmaceutical protease is intended to be used, catalytic activity is required at different locations in the body. Intended application matrices for pharmaceutical proteases are human or animal body fluids or cytoplasm of cells. The term “body fluid” is not limited to fluids in the strict sense but to all kind of body matrices, such as mucosa, organelles or entire organs. Preferred body fluids include but are not limited to blood, blood serum, blood plasma, digestive fluids such as intestinal and gastric juice and mucosa, synovial fluid, interstitial fluid, mucosal fluid, peritoneal fluid, extracellular matrix, the eye, cerebrospinal fluid, the brain, different organs as well as epithelial and mucosal surfaces of the body and the intracellular space including cytoplasm or cellular organelles such as lysosomes, endosomes, endoplasmic reticulum, Golgi apparatus, nucleus and mitochondria.
Each of the different application matrices have its particular composition of inhibitors of the enzymatic activity and appropriate proteases with activity in these environments are generated by the method of the invention irrespective of the particular composition of the inhibitors. In a preferred embodiment the proteases are active and insensitive or less sensitive to inhibitors in the blood, synovial fluid or the extracellular matrix.
The compositions of substances that the proteases are contacted with comprise at least one activity-modulating substance or a mixture of several such substances. Activity-modulating substances can either reduce, enhance or otherwise change the catalytic activity of a protease, e.g. they act as inhibitors or activators of the protease, respectively. In a preferred embodiment of the invention the activity-modulating substances are inhibitors which reduce or eliminate the catalytic activity of the protease.
The mechanism of inhibition is different for different inhibitors. Some inhibitors are competitive inhibitors, which reversibly bind to the protease. Other inhibitors bind irreversibly to the protease via a covalent bond or the inhibitors are irreversible by practical standards due to an extremely low binding constant. The invention provides a method for the selection of proteases with reduced inhibitor sensitivity independent of the mechanism of inhibition.
Protease Inhibitors in Different Matrices
Depending on the intended application matrix, the activity-modulating substances include but are not limited to carbohydrates, lipids, fats, polynucleic acids, peptides and proteins as well as all molecules belonging to the metabolism of the organism in which a therapeutic protease is intended to be used or any combination thereof. In a preferred embodiment the activity-modulating substances are polypeptide or protein inhibitors of the enzymatic function. In a more preferred embodiment the one or more activity-modulating substances are selected from the table 1 below.
In a most preferred embodiment the activity-modulating substances are protein inhibitors present in any part of the diseased body for which the protease is intended to be used. These inhibitors include but are not limited to protease inhibitors such as serpins, selected from the group consisting of alpha1-antitrypsin, alpha1-antichymotrypsin, kallistatin, protein C-inhibitor, leucocyte elastase inhibitor, plasminogen activator inhibitor, maspin, serpin B6, megsin, serpin B9, serpin B10, serpin B11, serpin B12, serpin B13, antithrombin, heparin cofactor, plasminogen activator inhibitor, alpha-2-plasmin inhibitor, C1-inhibitor, neuroserpin, serpin 12 and thyroxin-binding globulin; cystein protease inhibitors, selected from the group consisting of cystatin A, cystatin B, cystatin C, cystatin D, cystatin E/M, cystatin F, cystatin S, cystatin SA, cystatin SN, cystatin G, kininogen inhibitor unit 2 and kininogen inhibitor unit 3; metallo protease inhibitors, selected from the group consisting of TIMP-1, TIMP-2, TIMP-3 and TIMP-4; macroglobulins such as alpha2-macroglobulin; BIRC-1, BIRC-2, BIRC-3, BIRC-4, BIRC-5, BIRC-6, BIRC-7 and BIRC-8, and others. Other inhibitors are known to those skilled in the art (Rawling N D et al. Evolutionary families of peptidase inhibitors Biochemistry Journal (2004) 378: 705-716)
Substrates of Inhibitor-Sensitive Proteases
Either simultaneously or consecutively to the contact with the activity-modulating substance the protease variants are contacted with at least one substrate. The substrates include all substances amenable to chemical modification by a protease. These include peptides or proteins as present in the metabolism of an organism. In a preferred embodiment of the invention the substrate is a polypeptide or protein. In a more preferred embodiment the substrate is a protein whose function is relevant for the development of a disease or symptoms. In a most preferred embodiment the protein is a cytokine, such as APRIL, BAFF, BDNF, BMP, CD40-L, EGF, FasL, FGF, Flt3-L, Galectin-3, G-CSF, GM-CSF, IFN-alpha, INF-gamma, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, Leptin, LIGHT, Lymphotactin, M-CSF, MIF, NGF, Oncostatin-M, PDGF, RANKL, RANTES, TGF-alpha, TGF-beta, TNF-alpha, TNF-beta, TRAIL, or VEGF, or their respective receptors.
In addition the combination of at least one activity-modulating substance and at least one substrate may comprise any further number of substances which are neither activity-modulating nor substrate for the enzymatic activity (non-target molecules). These compositions include but are not limited to all substances of the application matrix of the protease or rather the entire application matrix which already comprises the at least one activity-modulating substance.
Compositions of Matrices for Screening
In a third step proteases with reduced sensitivity against the activity-modulating substances are selected. These proteases constitute the parent proteases for the generation of new libraries of protease variants that are subjected again to the selection process. Different compositions of activity-modulating substances are optionally contacted with the variety of proteases. These are sketched in
In a first embodiment of this step the concentration of the substances that the variety of proteases are contacted with in the step before is the same as the concentration of substances that are present in the application matrix. This embodiment can be applied when the parent protease has a residual activity that can reliably be measured in the presence of activity-modulating substances at the concentration of the application matrix. For example proteases are contacted with 100% serum, a substrate molecule and more active variants are selected. The complete method of the present invention when iteratively applied leads to variants with a higher activity in the presence of 100% serum than the starting protease.
In another embodiment the concentration of the activity-modulating substances is equivalent to the concentration of substances in the application matrix and thus the activity may be changed to a level that is outside the dynamic range of the assay format applied. This embodiment provides an approach applicable under these conditions. In such a case the protease variants are contacted with a dilution of the composition of substances in order to reduce the activity-modulating capacity of the composition to an extend that allows the activity of the proteases to be measured within the dynamic range of the assay. In a preferred aspect of this embodiment the dilution leads to concentration of the composition substances in the assay that corresponds to less than 100% of the concentration in the application matrix, more preferred to concentrations of 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.1% or less or any concentration in between and most preferred to concentrations from 70% to 5%. Improved variants that are selected at such at reduced concentration represent the basis for the generation of a new library of proteases. These proteases are subsequently contacted with a composition of substances at a concentration higher than the concentration applied to screen the parent proteases. In this iterative process the concentration of the composition of substances including the activity-modulating substances is increased stepwise in each round of the method. This generates proteases with gradually improved properties and allows screening to be performed under conditions where the residual activity of the proteases is within the dynamic range of the assay even in the presence of activity-modulating substances.
In a further embodiment the concentrations of all substances are increased beyond the concentration present in the application matrix, preferably to 101%, 110%, 120%, 150%, 200%, 300% or more or any concentration in between, more preferably from 120% to 200%. This embodiment provides a means to increase the selective pressure where the activity of proteases is measurable in the presence of 100% of the concentration of substances. In a preferred aspect of this embodiment the concentration of substances is increased stepwise over several cycles beyond the concentration of substances as they occur in the application matrix. In a more preferred aspect the concentrations of serum components in the assay correspond to 101%, 110%, 120%, 150%, 180%, 200%, 250% or 300% or any concentration in between. Most preferably the concentrations range from 120% to 200% of serum.
In the next embodiment the composition of substances present in the application matrix is selectively depleted of one or more of the activity-modulating substances while the concentrations of all other substances remain unchanged. The extend of depletion is adjusted in order to perform selection of proteases under conditions where the activity-modulating capacity of the composition of substances is reduced to a level that allows the enzymatic activity to fall within the dynamic range of the assay. In following rounds of the iterative optimization process the depletion of said components is reduced stepwise until the full concentration is reached. In a preferred aspect of this embodiment serum is depleted of protease inhibitors by one of several means including but not limited to standard chromatographic procedures such as affinity chromatography to reduce selectively the concentration of protease inhibitors. In one embodiment, molecules with high affinity for the inhibitor, the concentration of which needs to be lowered, are attached to a solid phase. These molecules include but are not limited to antibodies and proteases. In a next step the application matrix is contacted with the immobilized molecule, e.g. either in a batch mode, or a flow column. Alternatively, e.g. serum is incubated with a known amount of a serine protease such as trypsin, chymotrypsin, subtilisin or others which will react with and thereby reduce the concentration of inhibitors such as serpins, in particular alpha1-antitrypsin, antithrombin, antiplasmin and others. In successive rounds of optimization the amount of depletion of the activity-modulating substances is decreased stepwise, or it can be replenished at increasing levels.
In a further embodiment one or several of the components of the application matrix are enriched compared to the concentration of the application matrix. The relative enrichment is increased in successive rounds of screening to provide enhanced selection pressure. The enrichment factor is 101%, 110%, 120%, 150%, 180%, 200%, 250% or 300% of the concentration being present in the application matrix or any concentration in between. Preferred enrichment factors range from 120% to 200% of the concentration.
In a further embodiment the variety of proteases is contacted with isolated activity-modulating substances or a mixture thereof. The concentration of said activity-modulating substances are lower, equal or higher than the concentration of the respective substances in the application matrix. In a preferred aspect of this embodiment the activity-modulating substances are protease inhibitors. In a more preferred aspect the inhibitors applied are alpha1-anti-trypsin, anti-thrombin, anti-plasmin or alpha2-macroglobulin or any combination thereof. The concentrations vary from 200% down to 1% of the concentration present in serum.
Use of Display Technology for Screening
All of the embodiments detailed above may optionally include a pre-incubation step of the proteases with the activity-modulating substances for different lengths of time. In a preferred embodiment of the invention this pre-incubation time is 1 s, 1 min, 10 min, 30 min, 60 min, 2 h, 4 h, 6 h, 12 h, 24 h, 48 h, 72 h or longer, or any time in between. In a more preferred embodiment of the invention the pre-incubation time is 30 min, 60 min, 2 h or 4 h.
In another embodiment the population of proteases is displayed using phage panning. In order to identify proteases with altered sensitivity to activity-modulating substances, the library of phage particles is subject to incubation with one or more activity-modulating substances. Simultaneously or thereafter, the phage suspension is incubated with the substrate. Therefore, the substrate is coupled to a solid phase as is well known in the art for typical phage display targets, e.g. on latex beads, on living cells, in whole organisms or tissues, in multi-well plates, in “immuno-tubes”, on a column matrix or a number of other known formats. Such interactions rely on physico-chemical protein-surface interactions. The substrate can also be associated with other substances, but remain in the solution phase. Either type of interaction (solution or solid phase) rely on adsorption or on affinity interactions. There are a number of well known affinity interactions such as binding to specific antibodies, e.g. an anti-target antibody, or specific protein-protein interactions, e.g. antibody-protein A interactions. In a preferred aspect the affinity interactions are mediated by a peptide or chemical “tag” that is added to the target as a peptide fusion such as addition of a (his)6-tag, biotin binding peptide or a FLAG tag, or by post-translational conjugation such as addition of a biotin tag by chemical conjugation.
Phage particles presenting a protease which is insensitive or less sensitive to the activity-modulating substance, and possessing specificity for the substrate, bind to the substrate. Where the substrate is in solution (with or without additional associated substances), the substrate is then immobilized by direct capture of the target substrate or indirectly by capture of the associated substance. Where the substrate is bound to a solid phase before panning, this last immobilization step is unnecessary. Once the phage-substrate complex is immobilized, other phages that display more sensitive proteases or display no proteases or display proteases which are less specific, are depleted by washing. Typical washing include washing with solutions of adapted temperature and pH, containing low, medium or high salt, detergent, competitor protein or substrate, competitor phage, and other components. Washings are either done manually or automated, are done continuously or in discrete steps, and can involve changing the washing buffer composition as the washing progresses. The desired phages and thus the desired proteases are thus enriched on the solid phase. The phage is lysed and the encoding DNA recovered, e.g. by cloning or PCR amplification, or the phage is released from the solid phase, e.g., by acid washing, cleavage of the phage away from the protease or other means known in the art and used to infect a host cell for biological amplification. Other specialized selection schemes, such as the use of “selectively infective phage” where the substrate is bound to a ligand needed for host cell infection can also be used in the phage display enrichment of proteases with lowered sensitivity to activity-modulating substances.
In one aspect of this embodiment, protease variants, each with a defined mutation in the active site rendering the enzyme variants catalytically inactive, are displayed on a phage and selection is performed by panning on immobilised peptid substrates after incubation of the phages with the activity-modulating substances. In a more preferred embodiment such activity-modulating substances are protease inhibitors such as serpins, e.g. alpha1-antitrypsin, antithrombin and antiplasmin, or macroglobulins, e.g. alpha2-macroglobulin.
In another aspect of this embodiment one or more activity-modulating substances are covalently linked to a solid phase such as latex beads, living cells, whole organisms or tissues, multi-well plates, “immuno-tubes”, column matrix or other known solid phases. Then, the phage suspension is incubated with this activity-modulating substance, which preferentially binds phages presenting a protease variant susceptible to the activity-modulating substance and which preferentially leaves the insensitive or less sensitive variants unbound. In a further aspect of this embodiment the activity-modulating substances are left in the solution phase, either alone or associated with other substances in analogy to linkages and associations mentioned above. Then, the activity-modulating substances is immobilized by direct capture of the activity-modulating substances or indirectly by capture of the associated substance. Where the activity-modulating substance was bound to a solid phase before panning, this last immobilization step is unnecessary. The immobilization of the phage-activity-modulating substance complex will preferentially immobilize phage which display proteases that are more sensitive to activity-modulating substances. The non-bound phage, which are enriched for phage displaying proteases that are resistant to activity-modulating substances, are captured by recovering the fluid supernatant. In a preferred aspect this step is repeated once or several times. It is well known in the art that the density of the activity-modulating substances can be a critical parameter. Methods to modify the density or concentration of capture molecules are well known in the art. This “depletion” of undesired phage leaves the supernatant enriched for phage displaying proteases with lowered sensitivity to activity-modulating substances. The two aspects can be also combined, whereby a fraction of the activity-modulating substances is covalently linked and another fraction is left in the solution phase. The ultimately recovered phage are lysed and the encoding DNA recovered, e.g., by cloning or PCR amplification, or the phage are used to infect a host cell for biological amplification.
High-Throughput Screening of Protease Variants
Testing of the enzymatic properties is performed in a screening format where variants of the proteases are tested with respect to catalytic activity. In a preferred embodiment the screen is performed in a parallel high-throughput fashion in a miniaturized format in assay volumes less than 1 ml. In a more preferred embodiment the volume is less than 100 μl, for example 80 μl, 60 μl, 40 μl, 20 μl, or 10 μl or any volume in between. In an most preferred embodiment the (well-based) assay volume is less than 10 μl, for example 8 μl, 6 μl, 4 μl, 2 μl or 1 μl or any volume in between. In a further embodiment of the invention the screening volume is less than 1 μl, namely 800 nl, 600 nl, 400 nl, 200 nl, 100 nl or any volume in between.
In a preferred embodiment the coupling between phenotype and genotype is achieved by distributing individual cells of the transformed host into separated compartments. In a preferred embodiment the compartments are represented by the wells of a micro-titer plate. Variants of the enzymes are expressed in the compartments and contacted with activity-modulating substances and substrate. Activity is measured and variants with improved properties are selected.
Detection of the enzymatic activity is performed by measuring a physical change accompanied with the modification of the substrate. Changes introduced in the molecule by the modification include changes in activity, size, structure, composition, mass, reactivity, binding characteristics, or chemical properties such as solubility, acidity, color or fluorescence. A change in some of said properties can be measured indirectly by the incorporation of a chemical label into the substrate which changes its properties in response to the enzymatic conversion. In a preferred embodiment one or two fluorescent labels are covalently coupled to the substrate molecule. Substrate conversion is reflected in the change in one or several parameters of the fluorescence such as intensity, anisotropy, fluorescence lifetime, diffusion coefficient, fluorescence energy transfer, fluorescence intensity distribution, fluorescence coincidence analysis or cross-correlation. In a more preferred embodiment the substrate is covalently coupled with a fluorescent label in such a way that proteolytic cleavage leads to a change in the fluorescence anisotropy (EP 1307482). In a most preferred embodiment the proteolytic cleavage of the substrate is monitored by the accompanied loss of biological activity in a cell-based assay. In another preferred embodiment of the invention detection of cleavage of the substrate is performed by separation and detection of proteolytic fragments by chromatography, such as HPLC.
In another embodiment of the invention the method further comprises the step of selecting for protease variants having substantially similar or higher specificity with regard to the substrate as compared to the parent protease(s). Alternatively the protease variants obtained in step (c) may be modified as to exhibit a catalytic activity of defined specificity, whereby said defined specificity is not or only to a smaller extent occurring in the parent protease and/or the protease variants obtained in step (c). By such additional steps proteases are provided which have a defined specificity for therapeutic, research, diagnostic, nutritional, personal care or industrial purposes. Defined specificity means that the proteases are provided with specificities that do not exist in naturally occurring proteases. The specificities can be chosen by the user so that one or more intended target substrates are preferentially recognized and converted by the proteases. The specificity of proteases, i.e. their ability to recognize and hydrolyze preferentially certain peptide substrates, can be expressed qualitatively and quantitatively. Qualitative specificity refers to the kind of amino acid residues that are accepted by a protease at certain positions of the peptide substrate. For example, trypsin and t-PA are related with respect to their qualitative specificity, since both of them require at the P1 position an arginine or a similar residue. On the other hand, quantitative specificity refers to the relative number of peptide substrates that are accepted as substrates by the protease, or more precisely, to the relative kcat/kM ratios of the protease for the different peptides that are accepted by the protease. Proteases that accept only a small portion of all possible peptides have a high specificity, whereas the specificity of proteases that, as an extreme, cleave any peptide substrate would theoretically be zero. WO 2004/113521 provides a method for the generation and identification of proteases with desired specificities based on the combination of a protease scaffold that provides the general catalytic activity with variable specificity determining regions (SDRs) which provide the basis for the discrimination between different targets. In addition, the proteases can be fused either on DNA level or chemically to a binding module, e.g. a receptor fragment, an antibody domain or a fragment thereof, to address the target molecule. Furthermore, different mutagenesis methods can be employed to engineer specific proteases, e.g. single or multiple site-directed or random mutagenesis or transfer of amino acid residues or sequence stretches from one protease sequence to another. In another approach the specificity is generated by rational design.
Before subjected to a further round of screening selected variants may optionally be characterized in more detail with respect to the improved properties. In a preferred embodiment of the invention the IC50 of the variant is determined by incubating it with a serial dilution of the composition of activity-modulating substances and measuring the residual activity.
Determination of the Primary Structure of Protease Variants
In a preferred embodiment of the invention, protease variants with the desired properties in terms of activity, inhibitor insensitivity or any other property are identified in a screening process as described above. The result of the screening process is a culture of a clone of the organism expressing the protease variant of interest. From this culture deoxyribonucleic acid (DNA) sequence coding for said protease variant can be extracted by standard molecular cloning techniques known to anyone skilled in the art (e.g. Sambrook, J. F; Fritsch, E. F.; Maniatis, T.; Cold Spring Harbor Laboratory Press, Second Edition, 1989, New York). Isolation and cloning of the gene into suitable vectors allows the determination of the sequence of the deoxyribonucleic acid and thereby the amino acid sequence of the encoded protease by standard techniques.
Description of Inhibitor-Insenstive Protease Variants
The scaffold of the parent protease preferably belongs to the class of S1-serine proteases. Preferably the protease is derived from a trypsin-like protease, more preferably is derived from a human trypsin such as human cationic trypsin, human anionic trypsin and human mesotrypsin, most preferably the protease is derived from human cationic trypsin with the amino acid sequence shown in SEQ ID NO: 5.
As set forth in (1) above the protease has one or more mutations at positions that correspond structurally or by amino acid sequence homology to the regions 18-28, 34-41, 46-68, 90-102, 110-120, 123-137 and 162-186, 195 and 214 in human cationic trypsin. It is preferred that the protease has one or more mutations at one or more positions selected from the group of positions that correspond structurally or by amino acid sequence homology to the regions 20-26, 36-39, 51-59, 63-67, 78, 92-99, 112-118, 124-128, 131-134, 172-184, 195 and 214 in human trypsin. It is more preferred that the protease has one or more mutations corresponding to the following positions in human trypsin: 21, 22, 23, 24, 28, 37, 39, 46, 52, 55, 56, 57, 64, 66, 67, 78, 92, 93, 98, 99, 112, 115, 118, 124, 125, 128, 131, 133, 163, 172, 174, 181, 183, 184, 195 and 214, and most preferred at one or more of the following positions 22, 23, 24, 37, 52, 57, 64 and 133.
Even more preferably the protease has one or more mutations at positions that correspond structurally or by amino acid sequence homology to the positions:
G at position 21 is substituted by A, D, S or V, preferably D or V;
Y at position 22 is substituted by T, H, Q, S, W, G or A, preferably by T or H;
H at position 23 is substituted by T, N, G, D, R or Y, preferably by T or N;
F at position 24 is substituted by I, V, Q, T, L or A, preferably by I or V;
S at position 28 is substituted by A;
S at position 37 is substituted by T;
G at position 39 is substituted by S;
I at position 46 is substituted by V, N, L or T, preferably by V;
E at position 52 is substituted by V or M, preferably by V;
N at position 54 is substituted by S;
I at position 55 is substituted by T, N or R, preferably by T or N;
E at position 56 is substituted by G or R, preferably by G;
V at position 57 is substituted by A, T or G, preferably by A;
F at position 64 is substituted by I or T, preferably by I;
N at position 66 is substituted by D;
A at position 67 is substituted by V;
R at position 78 is substituted by W;
S at position 92 is substituted by T;
R at position 93 is substituted by P;
A at position 98 is substituted by D;
R at position 99 is substituted by H;
T at position 112 is substituted by A or P, preferably by A;
K at position 115 is substituted by M;
I at position 118 is substituted by V;
T at position 124 is substituted by K or I, preferably by K;
A at position 125 is substituted by P or S, preferably by P;
G at position 128 is substituted by R, K or T, preferably by R;
Y at position 131 is substituted by F, N or H, preferably by F;
D at position 133 is substituted by G;
V at position 163 is substituted by A;
S at position 172 is substituted by T;
Q at position 174 is substituted by R;
V at position 181 is substituted by A;
C at position 183 is substituted by H, Q or R, preferably by H;
N at position 184 is substituted by K or D;
D at position 195 is substituted by E; and/or
K at position 214 is substituted by E, D, R, T, or V, preferably by E
Most preferable is a trypsin mutant of SEQ ID NO:5 having one of the following combination of mutations:
S37T, E52V, E56G, V57A, F641, R78W, D133G, C183H (variant A); or
Y22T, H23T, F24I, S37T, E52V, E56G, V57A, F64I, R78W, D133G, C183H (variant B); or
Y22H, F24V, S37T, E52V, E56G, V57A, F641, R78W, D133G, C183H (variant C); or
Y22T, H23T, F24I, S37T, E52V, 155N, E56G, V57A, L58A, E59Q, F64T, R78W, R93P, T124K, A125P, G128R, Y131H, D133G, L135V, D139N, V163A, C183H, D195E, D214E (variant E); or
G21D, Y22T, H23T, F24I, S28A, S37T, E52V, N54S, 155T, E56G, V57A, F64I, R78W, R93P, R99H, T124K, A125P, D133G, V163A, C183H, D195E, K214E (variant F); or
G21V, Y22T, H23T, F24I, S28A, S37T, E52M, N54S, 155T, E56R, V57A, F64I, R78W, S92T, R93P, A98D, R99H, T112A, T124K, A125P, D133G, V163A, S172T, C183Q, D195E, K214E (variant G); or
G21D, Y22T, H23T, F24I, S28A, S37T, G39S, 146T, E52M, N54S, 155T, E56G, V57A, F64I, A67V, R78W, S92T, R93P, A98D, R99H, T112A, K115M, I118V, T124K, A125P, D133G, V163A, S172T, V181A, C183Q, N184D, D195E, K214E (variant D);
The numbering in all of the described mutations refers to SEQ ID NO:5, i.e. human cationic trypsin. The variants are those prepared in the Experimental Section of the application.
Expression of Protease Variants
In order to express the proteases of the invention, the DNA encoding such protease is ligated into a suitable expression vector by standard molecular cloning techniques (e.g. Sambrook, J. F; Fritsch, E. F.; Maniatis, T.; Cold Spring Harbor Laboratory Press, Second Edition, 1989, New York). The vector is introduced in a suitable expression host cell that expresses the corresponding protease. Particularly suitable expression hosts are bacterial expression hosts such as Escherichia coli, Pseudomonas fluorescence or Bacillus subtilis, or yeast expression hosts such as Saccharomyces cerevisiae, Kluveromyces lactis, Hansenula polymorpha or Pichia pastoris, other fungal expression hosts such as Aspergillus niger or Trichoderma reesei or mammalian expression hosts such as mouse (e.g., NS0), Chinese Hamster Ovary (CHO) or Baby Hamster Kidney (BHK) cell lines, transgenic mammalian systems such as rabbit, goat or cattle, other eukaryotic hosts such as insect cells or viral expression systems such as bacteriophages like M13, T7 phage or Lambda, or viruses such as vaccinia and baculovirus expression systems.
Often, the DNA is ligated into an expression vector behind a suitable signal sequence that leads to secretion of the protease into the extracellular space, thereby allowing direct detection of enzyme activity in the cell supernatant. Particularly suitable signal sequences for Escherichia coli, other Gram negative bacteria and other organisms known in the art include those that drive expression of the HlyA, DsbA, PhoA, PelB, OmpA, OmpT or M13 phage GIII genes. For Bacillus subtilis, particularly suitable signal sequences include those that drive expression of the AprE, NprB, Mpr, AmyA, AmyE, Blac, SacB, and for S. cerevisiae or other yeast, include the killer toxin, Bar1, Suc2, Matx, Inu1A or Ggpip signal sequence.
Alternatively, the enzyme variants are expressed intracellularly. As an alternative, after intracellular expression of the enzyme variants, or secretion into the periplasmatic space using signal sequences such as those mentioned above, a permeabilisation or lysis step is used to release the protease into the supernatant. The disruption of the membrane barrier is effected by the use of mechanical means such as ultrasonic waves, French press, cavitation or the use of membrane-digesting enzymes such as lysozyme.
As a further alternative, the genes encoding the protease are expressed cell-free by the use of a suitable cell-free expression system. For example, the S30 extract from Escherichia coli cells is used for this purpose as described by Lesly et al. (Methods in Molecular Biology 37 (1995) 265-278). In cell-free systems, the gene of interest is typically transcribed with the assistance of a promoter, but ligation to form a circular expression vector is optional. Regardless of the presence of a circular vector or the final host organism, the DNA sequence of the protease expression construct is determined using techniques that are standard in the art.
Purification of Protease Variants
As described above, the identified proteases are expressed in a variety of expression systems and the appropriate down-stream processing and purification procedures are selected accordingly. In a preferred embodiment of the invention the protease variant is expressed in a microbial host and the protein is secreted into the periplasmic or extracellular space. Cells carrying an appropriate expressing construct for the protease variants may be preserved as cryo stocks, well known to anyone skilled in the art. Cultures for protein expression are inoculated from a cryo stock and the volume of the culture increased successively in the appropriate container. In a preferred embodiment the cells are grown in a fermenter under controlled conditions of pH, temperature, oxygen and nutrient supply. After harvesting a first step comprises the separation of cells from supernatant using one or more of several techniques, such as sedimentation, microfiltration, centrifugation, flocculation or other. In a preferred embodiment the method applied is microfiltration.
In a preferred embodiment of the invention the protein is secreted into the supernatant and a further step of purification comprises the concentration of the supernatant by ultrafiltration. Protein purification from the supernatant or concentrated supernatant is performed with one or more of several preferred chromatographic methods including but not limited to ion-exchange, hydrophobic interaction, hydroxyapatite, size fractionation by gel-filtration and affinity chromatography or any combination thereof. In a more preferred method the protein is purified by combining several ion-exchange chromatographic steps to obtain a high purity protein. An even more preferred method comprises the combination of a cation-exchange and an anion-exchange chromatography, optionally combined with further cation or anion-exchange chromatographies. An appropriate purification method yields a purity of the protein of >50%, in a more preferred method the purity is >80%, in an even more preferred method the purity is >90%, in a yet more preferred method the purity is >95% and in a most preferred method the purity is >98%.
Derivatives of Protease Variants
To further improve the properties of the protease for the intended application it may optionally be subjected to a variety of modifications which include but are not limited to:
a) fusion to a peptidic component, preferably being selected from, but not limited to the group consisting of binding domains, receptors, antibodies, regulatory domains, pro-sequences, serum albumin, or fragments or derivatives thereof, and/or
b) covalent conjugation to a natural or synthetic polypeptide or non-polypeptide moiety, preferably being selected from the group consisting of polyethylenglycols, carbohydrates, lipids, fatty acids, nucleic acids, metals, metal chelates, nano-particles, liposomes, dendrimers or fragments or derivatives thereof, and/or
c) introduction of consensus glycosylation sites into the protease variant that are glycosylated during the post-translational processing of the protease variant during biosynthesis and/or
d) introduction of one or more mutation, insertion, substitution or deletion that render the protease variant less sensitive to clearance of inactivation mechanisms such as, but not limited to, proteolytic degradation, induction of immunogenicity or receptor mediated cellular uptake and/or
e) incorporation into formulations and/or drug delivery devices for protection and slow release.
Use of Protease Variants
The protease variants selected by the method can be used in industrial, cosmetic, diagnostic or synthetic applications. A preferred application of the proteases is the use as therapeutics to reduce or cure the cause or symptoms of a disease which can be prevented or treated by a protease therapy. Indications where a protease therapy is beneficial for the patient include inflammation and autoimmune diseases, cancer, cardiovascular diseases, neurodegenerative diseases, allergies, host-versus-graft disease, bacterial or viral infections, metabolic disorders or any other diseases where a protease therapy is indicated. Preferred embodiments of the present invention comprise proteases with beneficial activity for the indications of cancer, inflammation and autoimmune diseases. A more preferred application is in chronic inflammation. Different types of arthritis, rheumatoid arthritis, osteoarthritis, Sjörgen's syndrome, systemic lupus erythematosus, ankylsing spondylitis, psoriasis, inflammatory bowel diseases, Crohn's disease and ulcerative collitis all belong to this area and even today there is a constant need for effective drugs to treat these conditions. In a particularly preferred embodiment the application is rheumatoid arthritis, inflammatory bowel diseases, psoriasis, Crohn's disease, Ulcerative colitis, diabetes type II, classical Hodgkin's Lymphoma (cHL), Grave's disease, Hashimoto's thyroiditis, Sjogren's Syndrome, systemic lupus erythematosus, multiple sclerosis, Systemic inflammatory response syndrome (SIRS) which leads to distant organ damage and multiple organ dysfunction syndrome (MODS), eosinophilia, neurodegenerative disease, stroke, closed head injury, encephalitis, CNS disorders, asthma, rheumatoid arthritis, sepsis, vasodilation, intravascular coagulation and multiple organ failure, as well as other diseases connected with hTNF-alpha.
Several combinations of the above described embodiments can be defined leading to particular useful variants of the method of the invention. It is understood that the 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. The invention is further illustrated by the following examples which should not be construed as limiting. The content of all publications, patents, and patent applications cited herein are hereby incorporated by reference.
Random mutagenesis is done by a variation of the standard PCR purification protocol. A PCR reaction is set up in total volume of 100 μl containing a final concentration of 10 mM Tris/HCl pH 8.3, 50 mM KCl, 0.01% (wt/vol) gelatin, 7 mM MgCl2, 0.5 mM MnCl2, 0.2 mM dATP and dGTP, 1 mM dCTP and TTP, 0.3 μM of primers P1 and P2, 5 fmol template DNA and 2.5 units Taq polymerase. The following PCR program is used: 95° C. 1:00/95° C. 0:30/68° C. 0:30/72° C. 1:00 with 30 PCR cycles from step four to step two.
Alternatively, mutagenesis at specific sites is done by another variation of the standard PCR protocol. Therefore, two sets of primers are used: primers P1 and P3 binding to 5′ and 3′ ends of the gene and primers P2 and P4 binding to the site that is to be mutagenited and containing at specific positions mixtures of the four nucleotides. For the first round of PCR, two separate reactions are set up in a total volume of 100 μl, each containing 1×PCR-Buffer (KOD-Puffer), 0.2 mM dNTPs, 2 mM MgSO4, 0.3 μM of each primer, 20 ng template DNA and 2.5 units KOD polymerase. The following PCR program is used: 94° C. 2:00/94° C. 0:30/55° C. 0:30/68° C. 0:45/6° C. for ever with 22 PCR cycles from step four to step two. To generate the first fragment, primers P1 and P3 are employed whereas primers P2 and P4 are used for the second fragment. Consecutively, the two PCR products are separated from remaining template DNA by preparative agarose gel electrophoresis and purified using a gel purification kit (Qiagen). The two PCR products are mixed in an equimolar ratio with a total amount of 100 μg and serves as a template for a extension reaction carried out in a reaction mixture essentially analogous to the one above, with the terminal primers P1 and P2.
2 μg of the generated PCR fragment are digested with restriction endonucleases. In a similar approach, 8 μg of a standard plasmid for introducing genes into Bacillus subtilis plasmid (Palva I. et al. Secretion of Eschirichia coli beta-lactamase from Bacillus subtilis by the aid of alpha-amylase signal sequence. Cell Biology (1982) 79:5582-5586) are cut with restrictionendonucleases and dephosphorylated with CIAP. The digest of the plasmid DNA is heated to 50° C. followed by phenol-extraction. Finally, the PCR fragment and the plasmid DNA are both purified. Ligation is carried out over night at 16° C. according to the manufacturer's instructions (MBI fermentas). After heat-inactivation at 65° C. for ten minutes the DNA is subjected to ethanol precipitation, dried and transformed into Escherichia coli cells. The transformed cells are suspended in LB medium and grown over night at 37° C. in a shake flask incubator. The plasmid DNA of the generated library is then purified and transformed into Bacillus subtilis according to the protocol of Spizizen (Spizizen J. Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc. Natl. Acad. Sci. US (1958) 44:1072-1978) for assessing the individual variants.
The principle lying behind the selection and screening strategy is illustrated in
In order to identify enzyme variants having the desired substrate specificity, a screening approach based on a confocal fluorescence spectroscopy set-up as disclosed in WO 9416313 was used. A cell suspension of a Bacillus subtilis in culture medium was dispensed at a cfu-concentration ensuring that single cells are dispensed in each well of the microtiter plate. Cultures are grown over night at 37° C. and protein is secreted into the supernatent. Serum is added in a dilution so that the final concentration in the assay allows detection of the enzymatic activity. After adding the substrate (TNF-alpha covalently labelled with a fluorophore) to the sample and incubation for a certain period of time, the samples were subjected to measurement by confocal fluorescence spectroscopy. If necessary, this procedure was repeated several times in order to measure kinetics of the proteolytic cleavage. Samples were ranked according to proteolytic activity, and samples exceeding a certain activity threshold were identified in order to isolate the gene encoding the corresponding protease variant. The distribution of proteolytic activities of protease variants at different concentrations of serum obtained by this procedure is shown in
The screening of a library that contained a diverse population of candidate protease enzymes, each individually harboured on the expression plasmid, pBSX-G3-Zero, resulted in specific candidates improved in the examined characteristics, e.g. inhibitor resistance. Since the individual members of the library were tested as individual cultures, the result of the screening process was the identification of specific cultures that express putatively improved protease genes. The best candidate culture was plated to result in single colonies on solid LB-agar plates containing neomycin for selection (20 μg/ml). A resulting isolated colony was inoculated into liquid LB medium containing neomycin for selection (20 μg/ml), and plasmid DNA was prepared by Zymoprep kit (Zymo Research). The resulting plasmid DNA was transformed into E. coli strain XL1-blue using standard methods, amplified by cell growth in Luria-Burtani solid and liquid medium supplemented with 20 micrograms/ml of neomycin and isolated by Qiagen kit. DNA sequencing reactions were generated using the resulting plasmid DNA with a commercial kit (GenomeLab DTCS Quick Start Kit, Beckman Coulter) and the improved protease gene sequence determined using the CEQ 2000XL DNA Analysis System (Beckman Coulter). The DNA sequence of a candidate improved protease gene was unambiguously determined by this method and the resulting DNA sequence of the protease gene, through application of the standard genetic code for nuclear genes (see Sambrook, J. F; Fritsch, E. F.; Maniatis, T.; Cold Spring Harbor Laboratory Press, Second Edition, 1989, New York), unambiguously determined the amino acid sequence of the encoded protease protein.
A more detailed characterization of the inhibitor sensitivity is obtained by the determination of IC50's of different variants. The IC50 is the concentration of inhibitor at which the residual activity is reduced to 50% of the uninhibited value. The enzymes are incubated at a concentration of 1.5 μg/ml in PBS/pH 7.4/0.1% Triton-X-100 with a serial dilution of human serum in the same buffer, yielding the indicated final concentration. Fluorescently labelled TNF-alpha is added and proteolytic cleavage is followed over time at 37° C. by measuring changes in fluorescence parameters. The residual activity is normalized to the uninhibited value and plotted against the serum concentration (
Human serum contains a large number of different protease inhibitors which cannot be differentiated in the experiment described in example 3. To identify inhibitors to which the enzymes are particularly sensitive these can be tested individually. The concentrations of the different inhibitors in the serum vary considerably, between 70 μg/ml for anti-plasmin, 180 μg/ml C1-inhibitor, 230 μg/ml anti-thrombin and 1.5 mg/ml for anti-trypsin and alpha2-macroglobulin. These concentrations are defined as 100% and the enzymes are incubated with dilution series' of the individual inhibitors. Fluorescently labelled TNF-alpha is added and proteolytic cleavage is followed over time at 37° C. by measuring changes in fluorescence parameters. The residual activity is normalized to the uninhibited value and plotted against the concentration of the inhibitors.
bacillus aminopeptidase I (Geobacillus/Bacillus stearothermophilus)
norwegicus)
sapiens)
fasciatus, Cerastes vipera, Ophiophagus hannah)
Sequence Listing, Free Text
| Number | Date | Country | Kind |
|---|---|---|---|
| 05104543.3 | May 2005 | EP | regional |
| Number | Date | Country | |
|---|---|---|---|
| 60685566 | May 2005 | US | |
| 60686021 | May 2005 | US |
