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WO2005087947A2 - Enzymes proteolytiques transgeniques presentant une specificite renforcee pour les recepteurs de cytokines - Google Patents

Enzymes proteolytiques transgeniques presentant une specificite renforcee pour les recepteurs de cytokines Download PDF

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WO2005087947A2
WO2005087947A2 PCT/US2005/008043 US2005008043W WO2005087947A2 WO 2005087947 A2 WO2005087947 A2 WO 2005087947A2 US 2005008043 W US2005008043 W US 2005008043W WO 2005087947 A2 WO2005087947 A2 WO 2005087947A2
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receptor
cytokine
proteases
protease
cytokine receptor
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WO2005087947A3 (fr
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Morin Gregg
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Meyer Pharmaceuticals Llc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1058—Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6489—Metalloendopeptidases (3.4.24)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6489—Metalloendopeptidases (3.4.24)
    • C12N9/6491—Matrix metalloproteases [MMP's], e.g. interstitial collagenase (3.4.24.7); Stromelysins (3.4.24.17; 3.2.1.22); Matrilysin (3.4.24.23)

Definitions

  • BACKGROUND Inflammatory events play a central role in the pathology of disease conditions that adversely affect a considerable proportion of the population in developed countries. This process is mediated by cytokines, a system of polypeptides that enable one cell to signal to initiate events in another cell that initiate inflammatory sequelae. Normally, the system acts as part of a defensive reaction against infectious agents, harmful environmental agents, or malignantly transformed cells. But when inflammation exceeds the requirements of its defensive role, it can initiate adverse clinical effects, such as arthritis, septic shock, inflammatory bowel disease, and a range of other human disease conditions. Small-molecule antirheumatic drugs such as methotrexate and sulfasalazine are insufficient to control inflammation in about two-thirds of arthritis patients.
  • a leading biological agent for treating inflammatory conditions is Enbrel ® (Etanercept), marketed by Amgen Corp. It is a chimeric molecule comprising the extracellular portion of the human TNF receptor linked as a dimer to the IgG Fc region.
  • Enbrel ® is licensed in the U.S. for treatment of patients with moderate to severe rheumatoid arthritis, juvenile rheumatoid arthritis, and psoriatic arthritis. Approval is expected in 2003 for treating ankylosing spondylitis. Sales of Enbrel ® were $750 million in 2001. Scaling up production to meet growing demand has been a challenge. The projected sales in the U.S. market for current indication is expected to reach $4 billion by 2005, just for current indications.
  • Enbrel ® The expected future revenue from Enbrel ® was key to the valuation of Immunex in its recent acquisition by Amgen Corporation for $10.4 billion.
  • Other biological agents currently licensed in the U.S. for treating arthritis are Remicade ® (Infliximab), a chimeric antibody that binds the TNF- ⁇ ligand; HumiraTM, a humanized anti-TNF- ⁇ antibody, and Kineret TM (Anakinra), a recombinant form of IL-1 Ra, an antagonist of the interleukin- receptor.
  • Remicade ® Infliximab
  • HumiraTM a humanized anti-TNF- ⁇ antibody
  • Kineret TM akinra
  • IL-1 Ra an antagonist of the interleukin- receptor
  • Receptors for the cytokines on the target effector cell are also released in certain inflammatory conditions (Gatanaga et al., Proc. Natl. Acad. Sci USA 87:8781- 8784, 1990; Brakebusch et al., J. Biol. Chem. 269:32488, 1994).
  • Gatanaga and Granger had isolated a polypeptide that causes the human TNF receptor (both the p55 and p75 isoforms) to be cleaved from the cell surface (U.S. Patent 6,569,664).
  • the enzyme can be used as an anti-inflammatory agent for treatment of septic shock, and proposed that it be used to treat other inflammatory conditions, such as arthritis, cachexia, and inflammatory heart disease.
  • Gatanaga and Granger isolated nine recombinant cDNA clones that encoded proteins implicated in TNF receptor release (U.S. Patent 6,593,456).
  • MP8 has proved to be effective in animal models for sepsis, edema, arthritis, multiple sclerosis, and allergic asthma.
  • Some subjects having inflammatory conditions do not respond to the medicaments currently available, and the consumer cost of existing biological agents can be over $10,000 per year. There is a need for new biological agents that work on different pathways and which can be produced for more modest cost.
  • This invention provides a system for producing pharmaceutical compounds useful for the treatment of inflammatory disease.
  • Naturally occurring proteases are subject to cycles of mutagenesis and specificity screening, in order to obtain proteases having novel sequences and a refined specificity for cleaving particular cytokine receptors. These proteases have improved efficacy and side effect profiles. They can be used to target specific cytokine pathways that have gone awry in certain disease conditions, and optionally tailored to the particular needs of individual patients.
  • One aspect of this invention is a method for producing a bioengineered cytokine receptor cleaving enzyme having improved specificity or reaction kinetics by directed evolution.
  • the method can involve obtaining one or more polynucleotides, each encoding a protease; producing an expression library by introducing mutations into the nucleotide sequence of the polynucleotide(s); expressing the library so that each member of the expression library can be selected according to the specificity of the encoded protein; contacting the proteases produced by the library with a target protease substrate; and selecting members of the library according to whether the encoded protein cleaves the target substrate. Exemplary techniques for carrying out each of these steps are illustrated in the description that follows.
  • Suitable targets of interest for the treatment of inflammatory disease include but are not limited to receptors such as the IL-1 type I receptor; IL-1 type II receptor, TNF p55 and p75 receptors, and receptors for IL-6, IL-8, IL-13, IL-15, IL-17, and IL-18.
  • the method for producing bioengineered cytokine receptor cleaving enzymes can involve any other techniques described in this disclosure.
  • a plurality of proteases can be tested for cleaving a first cytokine receptor containing a first cytokine cleavage site, and a second cytokine receptor, which is essentially identical to the first cytokine receptor except that it contains a second cytokine cleavage site in place of the first.
  • Enzymes that cleave both receptor isoforms will recognize conformation of the receptors it cleaves, instead of or in addition to recognition of primary sequence.
  • proteases that cleave a protein substrate in solution are transitioned to cleavage of cell surface cytokine receptors through an intermediate target, which is the original substrate in a membrane-bound form.
  • Selected proteases are then further evolved to cleave the native cell-surface receptor.
  • proteases that cleave a specific amino acid sequence are transitioned by placing the specific sequence into the cytokine cleavage site of the target receptor. Selected proteases are then further evolved back to the native receptor sequence.
  • such techniques are suitable mutatis mutandis not only for evolving cytokine receptor enzymes, but any enzymes specific for cell surface proteins.
  • Another aspect of the invention is a business method for producing a pharmaceutical composition useful in the treatment of inflammatory condition. Someone practicing the business method first chooses a target cytokine receptor that mediates the inflammatory condition in at least a proportion of the affected population.
  • a further aspect of the invention is a method for selecting proteases suitable for treating an inflammatory condition in a subject. The user first determines what cytokine(s) mediate the inflammatory condition in the subject, for example, by immunoassay on a fluid or tissue sample taken from the subject.
  • Bioengineered cytokine receptor cleaving enzymes or a combination thereof are selected specifically to match the subject's need by targeting receptors for each of the cytokines implicated in the pathology of the condition.
  • Use of receptor cleaving proteases as part of a tailored mixture in a therapeutic composition or treatment method is also described in this disclosure. A further understanding of these and other aspects of this invention will be apparent to the skilled reader from the description that follows, along with the appended claims.
  • DRAWINGS Figure 1 is a scheme for obtaining a protease optimized for cleavage of the TNF receptor p55 or p75 isoform (TNFR1 or TNFR2).
  • Expression vectors encoding proteases from the human genome are subject to several cycles of general mutagenesis and selection for substrate specificity. If needed, the specificity is further refined by several cycles of specific mutagenesis at or around the catalytic or substrate binding site. Selected clones are characterized by sequence and enzyme kinetics, and tested in cell-based receptor cleavage assays.
  • FIG 2 shows release of both the p55 and p75 isoforms of the TNF receptor by clone MP8, a model TNF receptor releasing protein As the TNF receptor is cleaved from the cell surface, it accumulates in the culture supernatant where it can be measured by ELISA.
  • Figure 3 shows the release of a panel of receptors from the surface of normal human monocytes. The protein showed specificity for release of receptors for the pro-inflammatory cytokines TNF (both receptor isoforms), IL-6, and IL-1 (type II receptor), but not for other cell surface proteins.
  • Figure 4 shows results of a septic shock experiment.
  • Sepsis was induced by injecting 10 ⁇ g LPS and 7 mg galactosamine intravenously into Balb/c mice.
  • MP8 is fully protective against LPS- induced septic shock, whether given simultaneously with the LPS challenge, or 3 hours in advance.
  • Figure 5 is taken from an experiment in which MP8 was tested for its ability to treat established disease in a collagen-induced animal model for arthritis. Affected animals were randomized on day 22, and treated daily with MP8 or saline control. There was a highly significant reduction in swelling in the affected joints of the MP8-treated groups compared with control (p ⁇ 0.001 ).
  • Figure 6 is taken from an experiment where MP8 was tested in an animal model for Multiple Sclerosis.
  • Figure 7 was obtained from an animal model for experimentally-induced Asthma. Mice were sensitized by immunizing with ovalbumin, and then challenged with the allergen in aerosol form. MP8 reduced the inflammatory sequelae, shown by fewer white blood cells migrating into the alveolar fluid, and a reduced proportion of eosinophils.
  • the optimized proteolytic enzymes of this invention are designed to advance the treatment of inflammatory disease by more precise targeting of cytokine pathways involved in the pathology.
  • This disclosure provides a set of proteases suitable for improved cleavage of any cytokine receptor.
  • Proteolytic cleavage of the target may inactivate the receptor, preventing transduction of the cytokine signal into the cell expressing the receptor.
  • the cleaved receptor is also released from the cell, and may have the capacity to bind and neutralize a molar equivalent of incoming ligand. This provides the protease with the potential for a dual mode of action.
  • the catalytic role of the protease means that a mole of administered protein has the ability to affect many moles of cytokine receptors.
  • the therapeutic effect per gram of administered compound has the potential to greatly exceed that which is currently achieved using biologicals that neutralize cytokine ligands or receptors in stochiometric amounts.
  • Some cytokine receptors like the TNF and IL-6 receptors, are cleaved and shed from the cell surface naturally — presumably, as part of a natural feed-back mechanism to dampen the inflammatory response.
  • the novel proteases of this invention are also designed to cause shedding of cytokine receptors.
  • TNF ligand which is synthesized as a membrane protein and cleaved by the metalloprotease TACE Cleavage of TNF ligand is a pro-inflammatory event Reduced cleavage of TNF ligand should improve the therapeutic ratio • More targeted cleavage
  • TNF shedding enzymes have multiple substrates An enzyme specifically designed to cleave a single receptor allows blocking of the target pathway with more surgical precision This allows the therapy to be targeted precisely to the underlying pathology, and should help minimize the increased susceptibility cytokine blocking biologicals create for opportunistic infections
  • cytokine receptor means any cell-surface protein that binds a cytokine as a regular ligand The receptor may transduce a signal into the cell upon binding the ligand, but this is not required to meet the meaning of the definition (e g , the IL-1 type II "decoy” receptor)
  • a cytokine receptor protease or cytokine receptor cleaving enzyme is by definition an enzyme that specifically cleaves one or more naturally occurring cytokine receptors on the cell surface.
  • the protease will have at least one of the following effects: inactivation of the receptor so that it no longer binds the cognate cytokine; inactivation of the receptor so that it no longer transduces a signal into the cell upon cytokine binding which it normally does; or release of the receptor from the cell in a form that may or may not be capable of binding ligand in the soluble form.
  • a cytokine receptor releasing protein is by definition any protein, polypeptide, or equivalent thereof that causes specific release or shedding of one or more naturally occurring cytokine receptors.
  • the protein may be a cytokine receptor releasing enzyme, or it may be a cofactor, regulator, transcription activator, or any other protein that has the effect of causing receptor release.
  • a “bioengineered” cytokine receptor cleaving enzyme or cytokine releasing protein is an enzyme or releasing protein which does not occur naturally in nature, but has been designed, selected, or manufactured for improved receptor specificity or enzyme kinetics according to the techniques described in this patent disclosure.
  • the terms "polypeptide”, “peptide” and “protein” are used interchangeably to refer to polymers of amino acids of any length, and their homologs and derivatives. They may be isolated from natural sources, or produced by recombinant expression or chemical synthesis. Unless otherwise indicated, the practice of the invention will employ conventional techniques of protein biochemistry, molecular biology, microbiology, recombinant DNA, immunology, and the use of medicaments in standard medical care.
  • Cytokines of particular interest include but are not limited to the following: • The p55 isoform of the TNF receptor (TNFR1), which binds both TNF ⁇ and TNF ⁇ (lymphotoxin). The p55 receptor is implicated as a pivotal signal transduction agent in several inflammatory conditions, such as arthritis. • The p75 isoform of the TNF receptor (TNFR2). • The IL-6 receptor, which plays a prominent role in particular inflammatory conditions, such as multiple sclerosis and osteoarthritis.
  • the IL-1 receptors (the target of IL-1 receptor agonist, KineretTM). Both the type I and type II receptors are suitable targets — even though the type II receptor doesn't transduce a signal, its release from the cell can neutralize incoming IL-1 and prevent activation of the type I receptor.
  • the IL-15 receptor especially the ⁇ -chain. IL-15 is believed to trigger IL-17 release. Soluble IL-15 receptor reduces arthritis in the CIA animal model.
  • the IL-17 receptor The IL-17 pathway is believed to mediate release of pro-arthritic agents IL-6, IL-8, GM-CSF, and prostaglandin PGE2.
  • IL-13 receptor The IL-13 pathway is thought to be a central mediator in allergic asthma.
  • IL-18 receptor Presence of IL-18 correlates with disease activity in rheumatoid arthritis and osteoarthritis, and can be formed by chondrocytes. It is potentiated by IL-12 and IL-15, and feeds forward to upregulate IL-1 ⁇ and TNF ⁇ .
  • novel proteases of this invention can be obtained using a strategy in which a protease or pool of proteases is selected to start, and then the proteases are evolved through one or more cycles of mutation events and specificity selection or screening for the desired substrate specificity.
  • the protease is counter-screened for unwanted specificity or undesirable characteristics such as immunogenicity, turnover, or instability. Positively selected enzymes can then be characterized by sequencing, and further functional evaluation in biological assays.
  • Starting protease selection In selecting the starting protease, the user has the option of starting with an enzyme that is specific for a different substrate, and altering its specificity, or starting with an enzyme that has a measurable activity for the intended substrate, and improving the specificity.
  • the induced mutations and conformational changes will be the only potential antigenic epitopes.
  • human Factor Xa, Thrombin, and tPA are preferred choices, along with other human proteases involved in the thrombogenic, thrombolytic, and complement-activating cascades.
  • cytokine receptor cleaving proteases are members of the ADAM ("a desintegrin and metalloprotease") class of human MMPs, of which TACE is a member; as well as other human proteins implicated in cell surface protein cleavage or inactivation (U.S. Patent 6,593,456).
  • An effective strategy in evolving a protease of this invention in a rapid and efficient manner is to start with a pool of proteases, allowing those having a degree of activity towards the intended substrate to be selected early in the process.
  • the starting pool may comprise members of one or more of the classes already referred to, which are either secreted from cells in the native form, or which have been truncated or engineered to remove any membrane spanning portion, so as to be stable in soluble (non-membrane-bound) form. Proteins that are naturally secreted from the cell in a non- glycosylated form may pose less risk of immunogenicity.
  • Prototype proteases can be based on previously known cytokine receptors, such as those described in U.S. Patent Nos. 6,569,664 and 6,593,456.
  • new naturally occurring cytokine receptor cleavage enzymes can be obtained by using the target cytokine receptor as bate in an antibody precipitation-based isolation of proteases secreted from inflammatory cells (e.g., THP-1 cells stimulated with PMA), or extracted from membranes or matrix of inflammatory cells.
  • Receptor cleaving enzymes can also be identified by comparing expression patterns (e.g., by microarray analysis) in conditions where receptor cleaving enzymes are expressed (e.g., during inflammation or cancer), compared with normal controls. Libraries of variants can then be made from such prototypes to be used in subsequent screening and selection process. It is also possible to use a prototype that has a high specificity for an unrelated substrate.
  • Enzymes that have high degrees of specificity for primary structure include the following: TEV protease, HIV-1 protease, BAR1 protease, Factor Xa, Thrombin, tissue- type plasminogen activator, Kex2 protease, TVMV-protease, RSV protease, MuLV protease, MPMV protease, MMTV protease, BLV protease, EIAV protease, SIVrnac protease, and IgA protease.
  • Other proteases with less refined specificity fall into the families of aspartyl, serine, threonine, and cysteine proteases, and the matrix metalloproteases (MMPs).
  • the amino acid sequence of the target cytokine receptor is reviewed for sequences recognized by proteases that are highly specific for a particular peptide. If such a sequence is found in a place that would cause inactivation of the receptor, then the corresponding protease is used as the prototype from which to evolve the bioengineered cytokine receptor cleaving enzyme, capable of accessing the specific sequence in the receptor structure.
  • the specificity of the enzyme can be evolved by substituting the recognized sequence into a conformationally accessible cleavage site in the cytokine receptor, selecting variants capable of accessing the site, and then weaning the protease through further cycles of evolution to recognize the native receptor sequence.
  • the starting protease pool is mutagenized to refine specificity by constructing an expression vector in which a nucleotide sequence encoding the enzyme is expressed under control of a suitable promoter that is active in the host cell or in vitro expression system used for screening.
  • the expression vector is then subject to one or more mutagenizing reactions that cause changes to be made in the nucleic acid, and hence into the encoded protein.
  • General mutagenesis over the full length of the encoded protease can be performed by DNA shuffling (Crameri et al., Nature 391 :288, 1998).
  • a pool of homologous proteins is fragmented and reassembled using cyclical DNA amplification to create a diverse pool of chimeric genes.
  • Alternative methods include the recombinant chain reaction (WO 01/34835), random priming recombination (WO 98/42728), the staggered extension method (WO 98/42728), and others (Ostermeier et al., Nature Biotech 17:1205, 1999). These methods can be combined with error-prone PCR to create additional sequence diversity (Cadwell et al. , PCR Methods Appl. 2:28, 1992).
  • specific mutagenesis methods can be used to introduce mutations into a confined region of the protease, such as in the catalytic or substrate binding site.
  • cassette mutagenesis method degenerate or mutant oligonucleotides are synthesized in vitro and used to replace part of the encoding region (Oliphant et al., Gene 44:177, 1986; Horwitz et al., Genome 31 :112, 1989).
  • active site or substrate contacting residues are mutated by site-directed mutation methods (e.g., by PCR amplification with mutagenized primers).
  • Alanine scanning mutation can be used to identify specific amino acids that mediate substrate activity or recognition.
  • yeast cells are engineered to be dependent on membrane localization of human Ras protein, which is bridged by the target sequence to a myristoylation signal.
  • This system can detect proteases with weak activity, since the Ras signal is amplified through a signal tra ⁇ sduction pathway.
  • a bacteria-based selection system (Sices & Kristie, Proc. Natl. Acad. Sci. USA 95:2828, 1998) has the target sequence incorporated in the bacteriophage ⁇ cl repressor. When the sequence is cleaved, lytic replication occurs, releasing phage containing the active protease. Screening can be done more directly by directly measuring the enzymatic activity of the protease or protease pool.
  • the expression library may be designed to cause secretion of the expressed protein into the medium.
  • the medium can then be combined with the target sequence labeled on opposite ends with a fluorescent quench pair (Example 2).
  • the target peptide sequence bridges two proteins that are themselves fluorescent. Cleavage of the sequence causes a measurable change in fluorescence signal overlap.
  • any susceptible region of the cytokine receptor can be used as the target peptide sequence.
  • Full sequences of representative human receptors is provided later in this disclosure (SEQ. ID NOs:1 to 18) .
  • Suitable targets for some receptors include the observed cleavage site for the natural shedding process: QIEN/VKGT (SEQ. ID NO: 19) for the p55 TNF receptor; QPGA/VHLP (SEQ. ID NO:20) for the p75 TNF receptor; and LPVQ/DSDD (SEQ. ID NO:21) for the IL-6 receptor.
  • the cleavage sit recognized by the bioengineered enzyme need not be constrained to the site used by naturally occurring proteases, of course, since any cleavage which releases receptor from the cell or otherwise prevents signal transduction will be effective in inactivating the cytokine signaling.
  • susceptible regions can be mapped by subjecting full-length or cell-surface receptor to limited cleavage with a relatively non-specific protease such as trypsin, papain, subtilisin, or pronase. The receptor is then analyzed for new N' and C terminal amino acids created by the cleavage.
  • Migration of a sequence specific protease from one specificity to another can be facilitated by combining with the target sequence at low concentration in the cleavage assay, or by using intermediate sequences in which only some residues are modified per iteration (WO 03/095670, Direvo Bio Tech AG).
  • the prototype enzyme normally cleaves substrate in solution
  • the enzyme can be evolved by first adapting it to recognize the same substrate when expressed from a cell surface. Subsequent evolution can then transition the specificity of the enzyme from cell-anchored prototype substrate, to the target cell- surface cytokine receptor.
  • the specificity of the proteases can also be negatively screened for undesired cross- reactivity for other substrates: for example, cleavage of membrane-bound TNF ligand (natural cleavage site LAQA ⁇ /RSS, SEQ. ID NO:22); critical cell-surface proteins such as CD4 or the insulin receptor; or other cytokine receptors that are wanted to be kept separate.
  • the selection and screening processes described so far are based primarily on the primary structure (amino acid sequence) of a peptide which is the intended cleavage site within the whole cytokine receptor molecule.
  • specificity of natural cytokine receptor proteases is believed to depend at least in part on conformational features (tertiary structure).
  • the effectiveness of a newly mutagenized protease targeted towards a cell surface cytokine receptor may be influenced by the conformational accessibility of the intended cleavage site. For this reason, screening for specificity of the enzyme at the level of tertiary structure can be conducted as well or instead of screening for amino acid sequence specificity.
  • One such method is to transduce the expression library into a host mammalian cell that expresses the target cytokine receptor naturally, or has been genetically engineered to express the receptor at an elevated level (U.S. Patent 6,569,664). Cloned cells can then be screened on the basis of density of the receptor, for example, using fluorescent labeled antibody. Cells that express an effective protease can be identified by a lower receptor density, and the cloned protease can be recovered.
  • the protease library can be taken through as many rounds of mutation and selection or screening as is necessary to obtain the desired specificity.
  • the user then has the option of sequencing the gene of selected clones, and deducing the amino acid sequence of the protease.
  • the evolved sequence can then be compared with the prototype sequence from which it was derived.
  • the user may wish to select out clones that have evolved undesirable sequence features —such as a change in glycosylation sites, disulfide linkages, or introduction of an immunogenic T cell epitope, as is evident in the new amino acid sequence.
  • the proteases can then be assessed for effectiveness by more sophisticated bioassays, such as cell surface cleavage.
  • Preparing biological agents for clinical testing also typically involves validation in one or more animal models for inflammatory disease. Accordingly, the selection process used during evolution of the protease should be designed to take into account the nature of animal modeling subsequently to be performed.
  • evolution of the proteases has been driven in large part towards specificity for the amino acid sequence of the cleavage site, animal testing may be constrained, since exact amino acid sequences are not shared amongst all species homologs in all mammals.
  • the user has several options: 1.
  • a protease selected using human sequences can be tested in non-human primates, which will typically have a sequence on the native cytokine receptors very close to the human sequence.
  • the protease evolution can be parallel tracked using mouse target sequences and human target sequences in parallel screening.
  • Proteases selected by mouse targets may then provide a model for proteases selected by exactly the same criteria using human targets.
  • a protease selected using human sequences can be tested in an animal which is transgenic for the human sequences.
  • the gene for the target human cytokine receptor can be substituted into the genome by homologous recombination to knock out the endogenous mouse homolog gene.
  • This knock-in mouse will have an intact cytokine-regulated inflammatory system, with the exception that the target cytokine receptor is susceptible to enzymes specific for the human sequence.
  • An alternative strategy is to plan the protease evolution screening process so that it selects enzymes that recognize or are specific for the tertiary (conformational) structure of the target receptor.
  • receptors in a normal mouse will be susceptible to cleavage by an enzyme also specific for the human receptor, to the extent that the two species orthologs adopt the same conformation.
  • Recognition of conformational structure will be enhanced when the selection procedure includes screening for cleavage of full-length receptor, particularly when expressed on the cell surface.
  • a particularly effective strategy to get cross-reactive proteases is by simultaneous double- screening using both species orthologs. For example, proteases expressed from a library of encoding sequences are screened for the ability to cleave both the human and the mouse full-length orthologs.
  • the proteases are screened for the ability to cleave both the native human cytokine receptor, and a recombinant variant in which the cleavage site for the mouse ortholog is substituted in place of the human site.
  • Proteases determined to be capable of cleaving both orthologs in a specific fashion recognize more than just the exact species cleavage site sequence — in other words, the shared conformational features. They are selected for further rounds of evolution. Screening with the two targets can be done simultaneously during rounds of mutation and selection. Alternatively, the evolution and screening can be done in a sequential fashion: the proteases are first screened and evolved to recognize and cleave the human sequence specifically.
  • the selected proteases are then screened and evolved to cleave the mouse ortholog, simultaneously verifying that the specificity for the human sequence is not lost. Since conformational features may be shared with other cell-surface receptors and other proteins, it may be useful to include in the screening system a negative selection for undesired cleavage activity, as described earlier — such as against TNF ligand, which is pro-inflammatory, or receptors involved in immune recognition. Thus, the bioengineered cytokine receptor cleavage enzyme will be selected to cleave both the human and mouse target cytokine receptor with high Km, while being relatively ineffective (i.e., having a low Km) for other cell surface receptors and ligands.
  • compositions and their use An important utility of the modified proteases of this invention is to affect signal transduction from cytokines in certain disease conditions. Products that promote receptor release have the effect of decreasing the density of a particular cytokine receptor on the surface of cells, which in turn decreases signal transduction from the cognate cytokine ligand. If the cytokine is implicated in the pathology of a disease condition, then administration of the protease to the subject should ameliorate the symptoms or long-term consequences of the condition.
  • Conditions for treatment Disease conditions contemplated for the clinical application of this invention include but are not limited to the following: • Arthritis. TNF, IL-6, IL-1 and other cytokines promote expression of nitric oxide synthetase, believed to be involved in disease pathogenesis of rheumatoid arthritis and other arthritis sub-types. • Heart failure. IL-1 ⁇ and TNF are believed to be central mediators for perpetuating the inflammatory process, recruiting and activating inflammatory cells. The inflammation depress cardiac function in congestive heart failure, transplant rejection, myocarditis, sepsis, and burn shock. • Crohn's disease.
  • the inflammatory process mediated by multiple cytokines leads to thickening of the intestinal wall, ensuing from lymphedema and lymphocytic infiltration. • Septic shock.
  • Non-limiting examples are multiple sclerosis, ankylosing spondylitis, psoriasis, psoriatic arthritis, osteoarthritis, arteriosclerosis, cachexia, ulcerative colitis, arteriosclerosis, inflammation brought on by microbial infection, and diseases that have an autoimmune etiology, such as Type I Diabetes, myasthenia gravis, and systemic lupus erythematosis.
  • the proteases of this invention that promote receptor cleavage activity can be administered with the objective of decreasing or normalizing cytokine signal transduction.
  • the polypeptide is given at regular intervals to lessen the inflammatory sequelae.
  • the treatment is optionally in combination with small-molecule anti- inflammatory agents (such as methyltrexate), or with other agents that affect signal transduction (such as cytokine blockers like Enbrel®, or receptor antagonists like Kineret®) or that lessen the extent of inflammation in other ways.
  • small-molecule anti- inflammatory agents such as methyltrexate
  • other agents that affect signal transduction such as cytokine blockers like Enbrel®, or receptor antagonists like Kineret®
  • Polynucleotides that encode the proteases of this invention can also be used to promote cytokine receptor cleavage by gene therapy.
  • the encoding sequence is operably linked to control elements for transcription and translation in human cells. It is then provided in a form that will promote entry and expression of the encoding sequence in cells at the disease site.
  • Forms suitable for local injection include naked DNA, polynucleotides packaged with cationic lipids, and polynucleotides in the form of viral vectors (such as adenovirus and AAV constructs).
  • Methods of gene therapy known to the practitioner skilled in the art will include those outlined in U.S. Patent Nos. 5,399,346, 5,827,703, and 5,866,696.
  • the ability to obtain receptor-specific proteases in the manner described in this disclosure can provide the clinician with a battery of different proteases targeted towards different cytokine pathways. This allows the clinician where appropriate to select a protease that is designed specifically for the diagnosed condition: for example, TNF p55 receptor cleaving enzyme for rheumatoid arthritis; IL-6 receptor cleaving enzyme for multiple sclerosis or osteoarthritis, IL-13 receptor cleaving enzyme for allergic asthma.
  • the use of cocktails of two or more proteases appropriate for particular conditions is also contemplated.
  • the individual protease or protease combination is tailored to the pathology underlying the condition in each patient.
  • the cytokine(s) implicated in a patient's condition is determined by taking a fluid or tissue sample from or around the disease site in the patient, and determining what cytokine ligands or receptors are present at a level substantially above normal. Determination of these levels can be accomplished by appropriate assay, such as immunocytochemistry, fluorescence activated cytometry, or enzyme-based immunoassay. The patent can then be administered with a protease specific for cleaving the receptor corresponding to the cytokine that has been determined to be at an elevated level in the sample.
  • a polypeptide or polynucleotide of this invention is typically prepared according to good manufacturing procedures acceptable to the responsible regulatory agency, and then purified away from other reactive or potentially immunogenic components present in the mixture in which they are prepared. Effective amounts of the compositions are usually determined by clinical criteria. The doses can sometimes be estimated by the amount required to cause receptor cleavage to a measurable extent; often by at least about 25%, more preferably by about 50% or 75%. An effective amount of the active ingredient is then compounded into a medicament in accordance with generally accepted procedures for the preparation of pharmaceutical preparations, such as described in the current edition of Remington's Pharmaceutical Sciences, Mack Publishing Co., PA.
  • Steps in the compounding of the medicament depend in part on the intended use and mode of administration, and may include sterilizing, mixing with appropriate non-toxic and non- interfering excipients and carriers, dividing into dose units, and enclosing in a delivery device.
  • the medicament will typically be packaged in a suitable container accompanied by or associated with written information about its intended use. The ultimate responsibility for patient care and the use of the compositions provided in this disclosure lies in the hands of the managing clinician.
  • Example 1 Illustration of directed evolution of a cvtokine-releasinq enzyme This example illustrates how the system embodied in this invention can be used to obtain an optimized enzyme that causes the TNF p55 receptor to be released from cells in vitro.
  • Figure 1 is a diagram of the algorithm upon which this illustration is based.
  • a group of starting proteases suitable for evolution is assembled by screening for proteolytic activity using in vitro TNF receptor releasing assays for either the p55 TNF receptor (TNFR1) or the p74 paralog (TNFR2).
  • the proteases are selected from proteolytic enzymes encoded in the human genome, according to the Unigene database. Proteases with at least 5% activity over background for the target receptor are included in the starting pool.
  • Candidates are first selected from the family of metalloproteases, and also from other proteases like TACE and elastase that have been reported to have a degree of cytokine receptor cleavage activity.
  • general mutagenesis is conducted by the DNA shuffling technique, enhanced by error-prone PCR.
  • the mutagenized protease gene pools are then cloned into a plasmid expression vector, and selected for specificity for the TNF p55 cleavage site QIEN/VKGT according to the Sices and Kristie method (supra).
  • Clones of mutagenized and selected enzymes are then cross-checked for desired primary structure specificity.
  • the encoded protein is expressed, purified, and then tested using the FRET assay described in Example 2.
  • Enzymes having the desired activity are then checked for their ability to cleave the intact receptor on the cell surface by measuring receptor release from THP-1 cells, normal human monocytes (Example 3), or cells recombinantly engineered to express TNF receptor at an elevated level (U.S. Patent 6,569,664).
  • Clones are selected that have specificity for the TNF p55 receptor, but are negative for other cell surface receptors, particularly TNF ligand. Enzymes having the desired specificity for p55 primary and tertiary structure of human TNF receptor are candidates for treating human inflammatory disease. They are sequenced to determine the residues that have been altered, to assess whether the changes are tolerable from an immunogenic standpoint. To model therapeutic administration, an orthologous starting enzyme pool from the mouse genome is taken through the same mutagenesis and screening using the corresponding mouse TNF receptor cleavage site. Clones are selected for primary and tertiary receptor structure in parallel with the methods used for the human enzymes. The selected clones can be tested for anti-inflammatory activity in mouse models for TNF mediated disease, illustrated in Examples 4 to 7.
  • Example 2 Measuring receptor cleaving activity by fluorescence resonance energy transfer Cytokine-specific proteolytic activity can rapidly be quantified by Fluorescence Resonance Energy Transfer (FRET).
  • FRET Fluorescence Resonance Energy Transfer
  • Peptides having the amino acid sequence of the TNF Receptor or other protein substrates are labeled at opposite ends with a fluorescence emitter and a fluorescence quencher.
  • the peptide is then incubated with a source of receptor cleaving enzyme, and fluorescence is measured.
  • the quenching group normally absorbs fluorescence from the emitter. But enzymatic cleavage of the peptide decouples the quenching group, and fluorescence emission increases proportionally.
  • a model protease designated MP8 was produced in an expression vector behind an N-terminal His Tag sequence followed by a thrombin site.
  • the protein extract was chromatographed on Q-Sepharose®, the peak was purified by fast-flow chromatography on Ni-NTA, and endotoxin levels was reduced on a Q-Sepharose® (HiTrap) column.
  • the peptides used as substrates in the cleavage assay were taken from the known sites of proteolytic cleavage of the whole protein.
  • the peptides were labeled with the fluorescence emitter (Edans-*) at the C-terminal, and the quenching hapten (Dabcyl- ⁇ ) at the N-terminal.
  • the assay is conducted in the presence of the metal cations Zn ++ (0.1 mM) and Ca ++ (2 mM), and corrected for cleavage in the presence of EDTA (20 mM).
  • Dependence on divalent cations confirms that the enzyme activity measured in this assay is a metalloprotease.
  • the assay mixture also contains a cocktail of protease inhibitors and bovine albumin. Cleavage is measured as the net change in fluorescence emission after incubating the enzyme with the peptide at 37°C for 3 hours.
  • Cytokine receptor peptides SE0 -- ID N0 : p55 TNF Receptor A-N-V-K-G-T-E-D-S-G-. 23 p55 TNF Receptor (peptide 2) A-K-G-T-E-D-S-G-T-T-* 24 p75 TNF Receptor A-C-T-S-T-S-P-T-R-* 25 IL-6 Receptor A-A-N-A-T-S-L-P-* 26
  • SE0 -- ID N0 pro TNF (TACE substrate) A-L-A-Q-A-V-R-S-S-S-R-* 27 TNO-211 (matrix metalloprotease A- ⁇ -Abu-P-Q-G-L-EO)-A-K-NH 2 28 substrate)
  • the model protease cleaved the peptides from both the p55 and p75 TNF receptors (TNF-R1 and TNF-R2).
  • the protease also cleaved the IL-6 receptor peptide with high activity.
  • the model protease also showed specificity for peptides taken from other receptors involved in the inflammatory pathway (specifically, the IL-6 receptor and IL-1 receptors), but not for control substrates spanning known cleavage sites of other proteases: MMP-1 is fibroblast collagenase, MMP-2 is stromalysin, MMP-3 is gelatinase A, MMP-11 is collagenase III, renin is an aspartate protease; malaria is a cysteine protease, and CMV is a serine protease.
  • Example 3 Measuring receptor cleaving activity by cell surface receptor release
  • the protein was incubated with THP-1 cells. This cell line expresses both the p55 TNF receptor and the p75 TNF receptor. After incubating for 45 minutes at 37°C, the cells were washed, immunostained for cell surface receptor, and counted by flow cytometry.
  • Figure 2 shows that MP8 cleaves both the p55 and p75 TNF receptors when presented on the surface of cells.
  • FIG. 3 shows the release of a panel of receptors from the surface of normal human monocytes.
  • the cells were incubated with model protein MP8, washed, and then stained with antibody specific for each of the enumerated cell surface proteins.
  • the protein showed specificity for release of receptors for the pro-inflammatory cytokines TNF (both receptor isoforms), IL-6, and IL-1 (type II receptor), but not for other cell surface proteins.
  • the CD28 antigen is implicated in second signaling of antigen presenting cells. The cleavage of this protein may convey benefits of reducing autoantibody levels that arise as a byproduct of inflammation. For this reason, it may be appropriate in some circumstances to tolerate (or even select for) cross-specificity for CD28.
  • Example 4 Animal model for septic shock A classic model for determining effectiveness of agents against cytokine-mediated inflammation is endotoxin-induced septic shock (Morrison et al., J Infect Dis 162:1063, 1990). When tested in this model, the prototype receptor releasing protein MP8 (TRRE) was found to be completely protective against septic shock in a dose-dependent fashion, whether given simultaneously with the LPS challenge, or 3 hours in advance.
  • TRRE receptor releasing protein MP8
  • MP8 U.S. Patent was prepared under contract by Alliance Protein Laboratories from source material produced at Biosource
  • the enzyme was purified using Nickel NTA column chromatography and Q-Sepharose® analytical column chromatography. Endotoxin level was reduced using
  • Example 5 Animal model for established arthritis Collagen-induced arthritis is a standard model for evaluating potential therapeutic agents for rheumatoid arthritis (Courtenay et al., Nature 283:666, 1980; Williams et al., Proc Natl Acad Sci USA
  • Arthritis was induced in 7-9 week old female DBA/1 LacJ mice by immunization with collagen on days 0, and 7, and then boosting with LPS on day 14. Treatment with MP8 was initiated on day 22 when arthritis was well established. This protocol effectively models rheumatoid arthritis as it is seen in the clinic, where patients are treated after the onset of inflammatory synovitis. At that time, animals with arthritis were randomized into three groups, and unaffected animals were excluded. The three affected groups were then treated for 18 consecutive days with saline control, or with MP8 at either of two different doses. Figure 5 shows the results.
  • Example 6 Animal model for multiple sclerosis Experimental Autoimmune Encephalomyelitis is an animal model for Multiple Sclerosis (Brown et al., Lab. Invest. 45:278, 1981).
  • Female SJL/J mice (6 weeks old) were randomized into 3 groups of 10.
  • Two mL of Myelin Proteolipid Peptide (PLP) was emulsified in 3 mL of Complete Freund's Adjuvant containing an additional 20 mg of M. tuberculosis H37Ra.
  • PLP Myelin Proteolipid Peptide
  • mice were immunized subcutaneously in the base of the tail and footpad with a total of 60 ⁇ g PLP. They were also given 400 mg pertussis toxin i.p. on days 0 and 2.
  • MP8 or saline control was administered s.c. every day from day -3 to day 20. Progression of the disease was measured up to day 21 on the following scale: 0 ⁇ normal; 1 ⁇ limp tail or hind limb weakness; 2 ⁇ both limp tail and hind limb weakness; 3 ⁇ partial hind limb paralysis; 4 ⁇ complete hind limb paralysis; 5 ⁇ moribund or sacrificed.
  • Figure 6 shows the results.
  • MP8 had four clinically important effects: It completely prevented the disease from appearing in a proportion of animals; it substantially delayed the onset of symptoms; it reduced the severity of the disease by over 3-fold; and the treated animals continued to show normal weight gain.
  • Example 7 Animal model for asthma This example illustrates an animal model of experimentally induced asthma.
  • mice were sensitized on Days 0, 7, and 14 with 10 ⁇ g ovalbumin in 1% aluminum hydroxide.
  • the mice were challenged with the allergen in aerosol form (5% wt/vol in saline).
  • Treatment with MP8 or control was administered 1 h before the aerosol challenge, and 24 h and 48 h afterwards.
  • lungs were lavaged under anesthesia with 2 x 0.5 mL buffer to recover cells in the alveolar fluid.
  • Figure 7 shows the results.
  • MP8 reduced the number of the white blood cells migrating into the alveolar fluid.
  • the proportion of eosinophils was also substantially reduced.

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Abstract

La présente invention concerne un système permettant de produire des nouveaux agents biologiques utiles pour le traitement de maladies inflammatoires. Les protéases d'origine naturelle sont soumises à des cycles de mutagenèse et de criblage de spécificité, afin d'obtenir des protéases présentant des nouvelles séquences et une spécificité avancée pour le clivage de récepteurs de cytokines particuliers. Les récepteurs tels que le récepteur TNF p55, et les récepteurs de IL-1, IL-6, IL-15, et IL-18 sont des cibles adéquates pour les protéases présentant une efficacité renforcée et des profils d'effets secondaires améliorés. Les nouvelles protéases obtenues selon le mode de réalisation décrit dans cette invention peuvent être utilisées pour traiter l'arthrite ainsi que d'autres affections inflammatoires lors d'une maladie, et ce, de manière spécifique pour chaque patient.
PCT/US2005/008043 2004-03-09 2005-03-09 Enzymes proteolytiques transgeniques presentant une specificite renforcee pour les recepteurs de cytokines WO2005087947A2 (fr)

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WO2007084342A3 (fr) * 2006-01-13 2007-10-25 Us Gov Health & Human Serv Il-15 et il-15r-alpha améliorées aux fins d'expression dans des cellules mammaliennes

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US6593456B1 (en) * 1996-11-06 2003-07-15 The Regents Of The University Of California Tumor necrosis factor receptor releasing enzyme
DE69738887D1 (de) * 1996-11-06 2008-09-18 Univ California Tumor necrosis factor rezeptor abspaltendes enzym, dessen zubereitungen und verwendungen
AU2001247206A1 (en) * 2000-02-25 2001-09-03 Biofocus Discovery Limited Methods and compositions for identifying a protease
AU2001245371A1 (en) * 2000-02-28 2001-09-12 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Regulators of type-1 tumor necrosis factor receptor and other cytokine receptor shedding
EP1361284A1 (fr) * 2002-05-10 2003-11-12 Direvo Biotech AG Procédé de préparation des protéases spécifique pour une séquence par evolution dirigée
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WO2007084342A3 (fr) * 2006-01-13 2007-10-25 Us Gov Health & Human Serv Il-15 et il-15r-alpha améliorées aux fins d'expression dans des cellules mammaliennes
US9303080B2 (en) 2006-01-13 2016-04-05 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, National Institutes Of Health Codon optimized IL-15 and IL-15R-alpha genes for expression in mammalian cells
US9725492B2 (en) 2006-01-13 2017-08-08 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Codon optimized IL-15 and IL-15R-alpha genes for expression in mammalian cells
US9790261B2 (en) 2006-01-13 2017-10-17 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Codon optimized IL-15 and IL-15R-alpha genes for expression in mammalian cells
US10428133B2 (en) 2006-01-13 2019-10-01 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Codon optimized IL-15 and IL-15R-alpha genes for expression in mammalian cells
US11339198B2 (en) 2006-01-13 2022-05-24 The United States Of America, As Represented By, The Secretary, Department Of Health And Human Services Codon optimized IL-15 and IL-15R-alpha genes for expression in mammalian cells

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