EP1735438A2 - Clivage de vegf et de recepteur vegf par des proteases de types sauvage et mutantes - Google Patents

Clivage de vegf et de recepteur vegf par des proteases de types sauvage et mutantes

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Publication number
EP1735438A2
EP1735438A2 EP05735778A EP05735778A EP1735438A2 EP 1735438 A2 EP1735438 A2 EP 1735438A2 EP 05735778 A EP05735778 A EP 05735778A EP 05735778 A EP05735778 A EP 05735778A EP 1735438 A2 EP1735438 A2 EP 1735438A2
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EP
European Patent Office
Prior art keywords
protease
granzyme
specificity
wild
proteases
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP05735778A
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German (de)
English (en)
Inventor
Sandra Waugh Ruggles
Jack Nguyen
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Gyre Therapeutics Inc
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Catalyst Biosciences Inc
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Application filed by Catalyst Biosciences Inc filed Critical Catalyst Biosciences Inc
Publication of EP1735438A2 publication Critical patent/EP1735438A2/fr
Withdrawn legal-status Critical Current

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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
    • 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
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6467—Granzymes, e.g. granzyme A (3.4.21.78); granzyme B (3.4.21.79)

Definitions

  • VEGF Vascular Endothelial Growth Factor
  • VEGFRs Three high affinity cognate VEGF receptors (VEGFRs) have been identified: VEGFR-l Flt-1, VEGFR-2/KDR, and VEGFR-3 Flt-4.
  • VEGFRs are cell surface receptor tyrosine kinases that function as signaling molecules during vascular development.
  • An observation common in pre-clinical studies of anti-angiogenic agents targeting VEGF has been potent and broad-spectrum inhibition of very diverse tumor types (solid tissue and hematological), which is consistent with the widespread dependence of cancer on angiogenesis irrespective of tissue of origin. Single i.v.
  • adenoviruses expressing soluble Flkl and Fltl transduce the liver, express high plasma levels, and sequester VEGF from its native receptors on endothelial cells.
  • These circulating VEGF receptors produce systemic inhibition of angiogenesis in corneal micropocket assays, and importantly produce strong and broad-spectrum inhibition of tumor angiogenesis and tumor growth in established lung, prostate, colon, brain and pancreas tumors in subcutaneous, orthotopic and transgenic models. See, e.g. Kuo et al. 2001 PNAS 98: 4605-10.
  • the efficacy of anti-angiogenic therapy has been demonstrated in a randomized phase III trial using the anti-VEGF monoclonal
  • PSSCL profiling is a proprietary technology that generates a complete substrate specificity profile or "fingerprint" of each engineered protease in a single assay.
  • the present invention provides compositions and methods for using proteases that cleave proteins known to be involved in disease.
  • proteases that cleave proteins known to be involved in disease.
  • wild-type and mutated granzyme B polypeptide (“mutein") polypeptides are provided that cleave VEGF or VEGF receptor, which is known to be involved in angiogenesis.
  • the resultant modified proteins are provided for use as agents for in vivo therapy of cancers and other angiogenesis-related pathologies, including but not limited to macular degeneration, inflammation and diabetes.
  • the invention also provides methods for the modification of proteases to alter their substrate sequence specificity, so that the modified proteases specifically cleave a VEGF or VEGF receptor protein. Cleavage of targeted VEGF or VEGFRs is provided for treatment of a broad range of cancers wherein the treatment results in reduction or inhibition of vascularization necessary for continued tumor growth.
  • this modified protease is a serine protease.
  • this modified protease is a mutein granzyme B or MT-SP1.
  • One embodiment of the invention involves generating a library of protease sequences to be used to screen for modified proteases that cleave VEGF or VEGFR at a desired substrate sequence.
  • each different member of the protease library is a protease scaffold with at least one mutation made to each member of the library.
  • the remainder of the protease scaffold has the same or a similar sequence to a wild-type protease.
  • the cleavage activity of each member of the library is measured using the desired substrate sequence from the VEGF or VEGFR target protein. As a result, proteases with the highest cleavage activity with regard to the desired substrate sequence are detected.
  • the number of mutations made to the protease scaffold is 1, 2-5 (e.g. 2, 3, 4 or 5), 5-10 (e.g. 5, 6, 7, 8, 9 or 10), or 10-20 (e.g. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20).
  • the mutation(s) confer increased substrate specificity.
  • the mutation(s) are positioned in the scaffold in at least one of the SI, S2, S3 and S4 sites.
  • the activity of the mutein protease is increased by at least 10-fold, 100-fold, or 1000-fold over the activity of the wild-type protease. In related aspects, the increase is in substrate specificity.
  • the members of the library are made up of randomized amino acid sequences, and the cleavage activity of each member of the library by the protease is measured.
  • This type of library is referred to herein as a substrate library.
  • Substrate sequences that are cleaved most efficiently by the protease are detected.
  • the substrate sequence in a substrate library is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids long.
  • the members of the substrate library are made up of randomized amino acid sequences, and the cleavage selectiveness of each member of the library by the protease is measured.
  • Substrate sequences that are cleaved most selectively by the protease are detected.
  • the substrate sequence in the substrate library is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids long.
  • the specificity is measured by observing how many different substrate sequences the protease cleaves at a given activity. Proteases that cleave fewer substrate sequences at a given activity have greater specificity than those that cleave more substrate sequences.
  • the substrate sequence is a part of the VEGF or VEGFR target protein.
  • the library peptides include the VEGF or VEGFR residues of the Pl, P2, P3 and P4 sites.
  • the efficiency of cleavage by the protease muteins of the invention of the detected substrate sequence is increased by at least 2-fold, at least 5-fold, at least 10-fold, at least 100-fold, or at least 1000-fold over the average activity of the library.
  • the sequence specificity of the MT-SPl muteins of the invention in cleaving the substrate sequence is increased by at least 10-fold, at least 100-fold, or at least 1000-fold over the cleavage activity of the of wild-type protease.
  • the invention provides a method for treating a patient having a VEGF or VEGFR-related pathology, such as cancer, macular degeneration, inflammation and diabetes.
  • the method involves administering to the patient a protease that cleaves a VEGF or VEGFR protein, so that cleaving the VEGF or VEGFR treats the pathology.
  • the treatment of cancer by administration of an engineered protease is in combination with treatment with at least one other anti-cancer agent.
  • the protease is an MT-SPl mutein. In another aspect of this embodiment, the protease is wild-type MT-SPl.
  • the patient having a pathology e.g. the patient treated by the methods of this invention, is a mammal, or more particularly, a human.
  • all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, controls.
  • the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the invention will be apparent from the following detailed description and claims.
  • FIG. 1 is a graphical representation of the results of the Pl-Asp-AMC positional scanning synthetic combinatorial library (PSSCL) and individual tetrapeptide kinetics for the grB 199 A, N218A, and Y174A variants.
  • PSSCL activity of each amino acid at the P2, P3 and P4 positions is displayed as a percentage of the activity of the amino acid at that position.
  • concentration of the granzyme B variant used was adjusted to match that of 50 nM wild-type granzyme B in the library (X for I99A, Y for N218 A, and X for Y174A), and the activity was measured over 1 hour.
  • FIG. 2 is a graphic representation of the Pl-Asp-AMC positional scanning synthetic combinatorial library (PSSCL) results highlighting the narrow P2 specificity of the I99R variant and the increase in P4-Leu preference for I99F granzyme B. Mutations mimicking homologous protease amino acids at position 99 alter P2 and P4 specificity.
  • FIG. 3 is a graphical representation of the results of the Pl-Asp-AMC positional scanning synthetic combinatorial library (PSSCL) for the N218A/R192A and N218A/R192E variants. Argl92 and Asn218 mutations in combination reduce the preference for acidic P3 amino acids.
  • the amino acids represented from left to right of each of the graphs' X-axes are Ala, Arg, Asn, Asp, Gin, Glu, Gly, His, He, Leu, Lys, Phe, Pro Ser, Thr, Trp, Tyr, Val and norleucine (Nle).
  • the y-axis reads from bottom to top: 0, 20, 40, 60, 80, 100.
  • FIG. 4 A is a graphical representation of the results of the Pl-Asp-AMC positional scanning synthetic combinatorial library (PSSCL) comparing mutein I99A N218 A granzyme B to wild-type to illustrate a broad profile at P4 and P3 positions and a narrow preference for P2-Phe, Tyr and Nor (i.e., norleucine, a nonnatural isostere of methionine). Note that the I99A/N218A granzyme B has dramatically altered extended specificity.
  • PSSCL Pl-Asp-AMC positional scanning synthetic combinatorial library
  • FIGS. 5A-5D are graphical representations of the PSSCL results for rat granzyme B.
  • the activity in each subsite well is normalized to 100%.
  • the three dimensional structure of rat granzyme B shown as a ribbon in FIG. 5E and as a surface in FIG. 5F.
  • the N-terminal side of the ecotin inhibitor's substrate-like binding loop is shown in medium gray (lower left residues), and the C-terminal side of the substrate is modeled in light gray (upper right residues).
  • FIG. 6 is a ribbon diagram of the replaced residues in CB06.
  • the CB06 mutein contains the modified residues I99A/ Y174E/N218A, as shown in Table 7.
  • FIG. 7 is a graphical representation of wild-type grB target specificity at the P2, P3 and P4 positions. An Asp residue at Pl is held constant.
  • FIG. 8 is a graphical representation of CB01 mutein grB target specificity at the P2, P3 and P4 positions. The CB01 mutein contains the modified residues I99A/ N218A, as shown in Table 7. An Asp residue at Pl is held constant.
  • FIG. 9 is a graphical representation of CB06 mutein grB target specificity at the P2, P3 and P4 positions. An Asp residue at Pl is held constant.
  • FIG. 9 is a graphical representation of CB06 mutein grB target specificity at the P2, P3 and P4 positions. An Asp residue at Pl is held constant.
  • FIG. 10 is a photograph of a protein gel depicting cleavage of VEGF-R2 by wild- type and mutein grB proteases.
  • FIG. 11 is a graphical representation of grB and grB-Fc activity representing successful secretion of these proteins from mammalian cells transfected with constructs encoding them, as compared to a control assay.
  • FIG. 12 is a graph of the percent cell proliferation of HUVEC against the loglO of the concentration of rat granzyme B or human granzyme B present to the culture.
  • FIG. 13A is a photograph of a protein gel depicting granzyme B found in serum of mice injected with adenovirus encoding granzyme B, as different numbers of days post-injection.
  • FIG. 13B is a graph showing the percent inhibition of angiogenesis in a microcorneal in vivo model.
  • FIG. 14 is a series of graphical representations of a PSSCL profile of wild-type and various mutants of granzyme B showing specificity of various muteins.
  • FIG. 14A depicts the PSSCL profile of of the granzyme B mutein Y151A (CB143).
  • FIG. 14B depicts the PSSCL profile of of the granzyme B mutein K97Y/I99A (CB125).
  • FIG. 14C depicts the PSSCL profile of of the granzyme B mutein I99A/N218A/R192Y (CB111).
  • FIG. 14D depicts the PSSCL profile of of the granzyme B mutein K97E/I99A/N218A Y174E (CB121).
  • FIG. 15 is a photograph of a SDS PAGE gel showing bands of MT-SPl purified by a one-column purification procedure and then re-folded through successive dialysis steps. MT-SPl variants were expressed in bacteria and purified from inclusion bodies. Each protease retains high catalytic activity and is >99% pure, and thus are making them appropriate for crystallographic studies.
  • FIG. 16A-H are a graphical representation of PSSCL profiles of wild-type MT- SPl and six variants.
  • FIGS. 16B-H are a graphic depiction of PSSCL profiles of MT-SPl muteins CB18 (FIG 16B), CB38 (FIG 16C), CB159 (FIG 16D), CB83 (FIG 16E), CB155 (FIG 16F), CB151 (FIG 16G), and CB152 (FIG 16H) showing narrowed specificity profiles.
  • the activity is represented in relative fluorescence units along the y-axis by dividing each amino acid activity by the activity of the best amino acid within each sublibrary.
  • FIG. 17 is a photograph of a protein gel showing VEGFR2-Fc is efficiently cleaved by wild-type and muteins of MT-SPl.
  • FIGS. 18A, 18B and 18C are graphic depictions of the PSSCL substrate specificity profile at P2, P3 and P4, respectively, of human MT-SPl in a Pl-Lys fixed library. The library format for each extended position is listed above the profile. The activity is represented in pM/sec on the y-axis for each amino acid along the x-axis.
  • FIG. 19 is a graphical representation of trypsin and MT-SPl protease activity over time in the presence of increasing levels of serum.
  • FIGS. 21 A is a graphical representation of the amount of proliferation of endothelial cells treated with increased concentrations of MT-SPl and the muteins CB18, CB83 and CB152.
  • FIG 7B is a photograph of a western blot showing the cleavage of VEGFR2 in HUNEC cells in the presence of MT-SPl, CB18 and CB83, respectively.
  • FIG. 7C is a graphical representation of the amount of soluble extracellular NEGFR2 released by HUVECs upon treatment with MT-SPl, CB18 and CB83.
  • FIG. 22 is a graphical representation of the maximum dose of MT-SPl, CB18 and CB 152 that can be tolerated by mice.
  • FIG. 23 is a graphical representation of the extent of inhibition of neovascularization by a dose of MT-SPl and CB18.
  • FIG. 24 is a graphical representation of the inhibition of vascular permeability by MT-SPl, CB18 and CB152 in the mouse Miles assay.
  • FIG. 25 is a photograph of a protein gel showing the cleavage of VEGF by wild- type MT-SPl but not the selective variant CB152. DETAILED DESCRIPTION OF THE INVENTION
  • Serine proteases have a highly adaptable protein scaffold. These proteases differ over a broad range in their substrate recognition properties, ranging from highly specific to completely non-specific. Despite these differences in specificity, the catalytic mechanism is well conserved, consisting of a substrate-binding pocket that correctly registers the scissile peptide in the active site. This large family of proteases can be broadly divergent among members in their sequence specificities yet highly conserved in their mechanism of catalysis. This is because substrate specificity is not only determined by local contacts directly between the substrate peptide and the enzyme (first sphere residues), but also by long range factors (second sphere residues). Both first sphere and second sphere substrate binding effects are determined primarily by loops between B-barrel domains.
  • subtilisin an enzyme with low specificity for hydrophobic residues at the Pl position, the authors of this reference managed to radically alter its specificity for tribasic residues radically by making 3 point mutations in the substrate binding pocket. The resulting mutant had over a 1000-fold specificity for tribasic substrates versus the original hydrophobic substrate. In total, studies on changing the specificity of proteases suggest it is possible to radically alter substrate specificity radically.
  • This invention discloses specific muteins of proteases having altered target specificity and methods for using them to treat disease.
  • This invention discloses specific muteins of the serine proteases having altered target specificity for VEGF or VEGFR.
  • Granzyme B is a serine protease (SI -type) necessary for target cell lysis in cell-mediated immune responses.
  • the wild-type protease cleaves after Asp in its consensus recognition site of I/V-E/Q/M-P/T-D (Table 11).
  • Granzyme B is linked to an activation cascade of caspases (aspartate-specific cysteine proteases) responsible for apoptosis execution.
  • caspases that Granzyme B cleaves include caspase-3, caspase-7, caspase-9 and caspase- 10 to give rise to active enzymes mediating apoptosis.
  • Membrane-type serine protease 1 (MT-SPl )/matriptase is an epithelial- derived integral membrane enzyme.
  • MT-SPl is a mosaic protein containing a transmembrane domain, two CUB domains, four LDLR repeats, and a serine protease domain.
  • the protease domain of MT-SPl has been expressed in bacteria or yeast in milligram quantities and purified.
  • PPSCL positional scanning substrate combinatorial libraries
  • allelic variant denotes any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence.
  • allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
  • complements of polynucleotide molecules denotes polynucleotide molecules having a complementary base sequence and reverse orientation as compared to a reference sequence.
  • the sequence 5' ATGCACGG 3' is complementary to 5* CCGTGCAT 3'.
  • degenerate nucleotide sequence denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp).
  • a "DNA construct” is a single or double stranded, linear or circular DNA molecule that comprises segments of DNA combined and juxtaposed in a manner not found in nature.
  • DNA constructs exist as a result of human manipulation, and include clones and other copies of manipulated molecules.
  • a "DNA segment” is a portion of a larger DNA molecule having specified attributes.
  • a DNA segment encoding a specified polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, which, when read from the 5' to the 3' direction, encodes the sequence of amino acids of the specified polypeptide.
  • expression vector denotes a DNA construct that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription in a host cell.
  • Such additional segments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like.
  • Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
  • isolated when applied to a polynucleotide molecule, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems.
  • isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones, as well as synthetic polynucleotides.
  • Isolated DNA molecules of the present invention may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78, 1985).
  • the term "isolated” indicates that the protein is found in a condition other than its native environment, such as apart from blood and animal tissue.
  • the isolated protein is substantially free of other proteins, particularly other proteins of animal origin. It is preferred to provide the protein in a highly purified form, i.e., at least 90% pure, preferably greater than 95% pure, more preferably greater than 99% pure.
  • operably linked when referring to DNA segments, denotes that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in the promoter and proceeds through the coding segment to the terminator.
  • ortholog denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.
  • polynucleotide denotes a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
  • Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules.
  • the length of a polynucleotide molecule is given herein in terms of nucleotides (abbreviated “nt”) or base pairs (abbreviated “bp”).
  • nt nucleotides
  • bp base pairs
  • nucleotides is used for both single- and double-stranded molecules where the context permits. When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term "base pairs”.
  • promoter denotes a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes.
  • a "protease” is an enzyme that cleaves peptide bonds in peptides, polypeptides and proteins.
  • a “protease precursor” or a “zymogen” is a relatively inactive form of the enzyme that commonly becomes activated upon cleavage by another protease.
  • secretory signal sequence denotes a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized. The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
  • substrate sequence denotes a sequence that is cleaved by a protease.
  • target protein denotes a protein that is specifically cleaved at its substrate sequence by a protease.
  • seraffold refers to a wild-type or existing variant protease to which various mutations are made. Generally, these mutations change the specificity and activity of the scaffold.
  • an existing variant protease is a protease existing in an organism which has been mutated at one or more positions compared to the wild-type protease amino acid sequence of the species to which the organism belongs.
  • an “isolated” or “purified” polypeptide or protein or biologically-active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of proteins in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly-produced.
  • the language "substantially free of cellular material” includes preparations of protease proteins having less than about 30% (by dry weight) of non-protease proteins (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-protease proteins, still more preferably less than about 10% of non-protease proteins, and most preferably less than about 5% of non-protease proteins.
  • non-protease proteins also referred to herein as a "contaminating protein”
  • the protease protein or biologically-active portion thereof is recombinantly-produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protease protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of protease proteins in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of protease proteins having less than about 30% (by dry weight) of chemical precursors or non-protease chemicals, more preferably less than about 20% chemical precursors or non-protease chemicals, still more preferably less than about 10% chemical precursors or non-protease chemicals, and most preferably less than about 5% chemical precursors or non-protease chemicals.
  • serine protease refers to proteases which contain the serine protease domain.
  • proteases include members of the serine protease family which are subdivided into structural subclasses (for example SI). Specific examples of serine proteases are provided below.
  • the term “selectiveness” or “Specificity” is a ratio of efficiency of cleavage of a targeted substrate site versus another substrate site that is not the targeted site.
  • the term “peptide” refers to a polypeptide of from 2 to 40 amino acids in length.
  • Protein scaffolds useful in the instant invention include unspecific and specific proteases or fragments of unspecific and specific proteases or are derived from unspecific or specific proteases.
  • derived from or "a derivative thereof in this respect and in the following variants and embodiments refer to derivatives of proteins that are mutated at one or more amino acid positions and/or have a homology of at least 70%, preferably 90%, more preferably 95% and most preferably 99% to the original protein, and/or that are proteolytically processed, and/or that have an altered glycosylation pattern, and/or that are covalently linked to non-protein substances, and/or that are fused with further protein domains, and/or that have C-terminal and/or N-terminal truncations, and/or that have specific insertions, substitutions and/or deletions.
  • derived from may refer to derivatives that are combinations or chimeras of two or more fragments from two or more proteins, each of which optionally comprises any or all of the aforementioned modifications.
  • the tertiary structure of the protein scaffold can be of any type. Preferably, however, the tertiary structure belongs to one of the following structural classes: class SI (chymotrypsin fold of the serine proteases family), class S8 (subtilisin fold of the serine proteases family), class SC (carboxypeptidase fold of the serine proteases family), class Al (pepsin A fold of the aspartic proteases), or class C14 (caspase- 1 fold of the cysteine proteases).
  • class SI chymotrypsin fold of the serine proteases family
  • class S8 subtilisin fold of the serine proteases family
  • class SC carboxypeptidase fold of the serine proteases family
  • class Al pepsin A fold of the aspartic prote
  • proteases that can serve as the protein scaffold of engineered proteases for the use as human therapeutics by cleavage of VEGF or VEGFR are or are derived from granzyme B, MT-SPl, human trypsin, human thrombin, human chymotrypsin, human pepsin, human endothiapepsin, human caspases 1 to 14, and/or human furin.
  • the protein scaffold has a tertiary structure or fold equal or similar to the tertiary structure or fold of the SI structural subclass of serine proteases, i.e.
  • the chymotrypsin fold and/or has at least 70% identity on the amino acid level to a protein of the SI structural subclass of serine proteases. It is preferred that amino acids are altered at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 18-25, 38-48, 54-63, 73- 86, 122-130, 148-156, 165-171 and 194-204 in human trypsin 1, and more preferably at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 20-23, 41-45, 57-60, 76-83, 125-128, 150- 153, 167-169, and 197-201.
  • the number of amino acid changes to be combined with this type of protein scaffold is preferably between 1 and 10, and more preferably between 2 and 4.
  • the protein scaffold is equal to or is a derivative or homologue of one or more of the following proteins: chymotrypsin, granzyme, kallikrein, trypsin, mesotrypsin, neutrophil elastase, pancreatic elastase, enteropeptidase, cathepsin, thrombin, ancrod, coagulation factor IXa, coagulation factor vlla, coagulation factor Xa, activated protein C, urokinase, tissue-type plasminogen activator, plasmin, Desmodus- type plasminogen activator.
  • the protein scaffold is trypsin or thrombin or is a derivative or homologue from trypsin or thrombin.
  • the trypsin or thrombin scaffold is most preferably of human origin in order to minimize the risk of an immune response or an allergenic reaction.
  • derivatives with improved characteristics derived from human trypsin are most preferably of human origin in order to minimize the risk of an immune response or an allergenic reaction.
  • the protein scaffold belongs to the S8 structural subclass of serine proteases andor has a tertiary structure similar to subtilisin E from Bacillus subtills-and o ⁇ has at least 70% identity on the amino acid level to a protein of the S8 structural subclass of serine proteases.
  • the scaffold belongs to the subtilisin family or the human pro-protein convertases.
  • amino acids are altered in the protein scaffold at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 6-17, 25-29, 47-55, 59-69, 101-111, 117-125, 129-137, 139-154, 158-169, 185-195 and 204-225 in subtilisin E from Bacillus subtilis, and more preferably at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 59-69, 101-111, 129-137, 158-169 and 204-225.
  • the protein scaffold is equal to or is a derivative or homologue of one or more of the following proteins: subtilisin Carlsberg; B.
  • subtilis subtilisin E subtilisin BPN'
  • B. licheniformis subtilisin B. lentus subtilisin
  • Bacillus alcalophilus alkaline protease proteinase K; kexin; human pro-protein convertase; and human furin.
  • Subtilisin BPN' or one of the proteins SPC 1 to 7 is used as the protein scaffold.
  • the protein scaffold belongs to the family of aspartic proteases and or has a tertiary structure similar to human pepsin.
  • the scaffold belongs to the Al class of proteases and/or has at least 70% identity on the amino acid level to a protein of the Al class of proteases.
  • amino acids are altered in the protein scaffold at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 6-18, 49-55, 74-83, 91-97, 112-120, 126-137, 159-164, 184-194, 242-247, 262-267 and 277-300 in human pepsin, and more preferably at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 10-15, 75-80, 114-118, 130-134, 186-191 and 280-296.
  • the protein scaffold is equal to or is a derivative or homologue of one or more of the following proteins: pepsin, chymosin, renin, cathepsin, yapsin.
  • pepsin or endothiopepsin or a derivative or homologue thereof is used as the protein scaffold.
  • the protein scaffold belongs to the cysteine protease family and/or has a tertiary structure similar to human caspase 7.
  • the scaffold belongs to the C14 class of cysteine proteases or has at least 70% identity on the amino acid level to a protein of the C14 class of cysteine proteases.
  • amino acids are altered in the protein scaffold at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 78-91, 144-160, 186-198, 226-243 and 271-291 in human caspase 7, and more preferably at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 80-86, 149-157, 190-194 and 233-238. It is preferred that the protein scaffold is equal to or is a derivative or homologue of one of the caspases 1 to 9.
  • the protein scaffold belongs to the SI 1 class of serine proteases or has at least 70% identity on the amino acid level to a protein of the SI 1 class of serine proteases and/or has a tertiary structure similar to D-alanyl-D-alanine franspeptidase from Streptomyces species Kl 5.
  • amino acids are altered in the protein scaffold at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 67-79, 137- 150, 191-206, 212-222 and 241-251 in D-alanyl-D-alanine franspeptidase from Streptomyces species K15, and more preferably at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 70-75, 141-147, 195-202 and 216-220 (numbering of amino acids according to SEQ ID NO: 15).
  • the D-alanyl-D-alanine franspeptidase from Streptomyces species K15 or a derivative or homologue thereof is used as the scaffold.
  • the protein scaffold belongs to the S21 class of serine proteases or has at least 70% identity on the amino acid level to a protein of the S21 class of serine proteases and or has a tertiary structure similar to assemblin from human cytomegalovirus.
  • amino acids are altered in the protein scaffold at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 25-33 64-69, 134-155, 162-169 and 217-244 in assemblin from human cytomegalovirus, and more preferably at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 27-31, 164-168 and 222-239. It is preferred that the assemblin from human cytomegalovirus or a derivative or homologue thereof is used as the scaffold.
  • the protein scaffold belongs to the S26 class of serine proteases or has at least 70% identity on the amino acid level to a protein of the S26 class of serine proteases and/or has a tertiary structure similar to the signal peptidase from Escherichia coli.
  • amino acids are altered in the protein scaffold at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 8-14, 57-68, 125-134, 239-254, 200-211 and 228- 239 in signal peptidase from Escherichia coli, and more preferably at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 9-13, 60-67, 127-132 and 203-209. It is preferred that the signal peptidase from Escherichia coli or a derivative or homologue thereof is used as the scaffold.
  • the protein scaffold belongs to the S33 class of serine proteases or has at least 70% identity on the amino acid level to a protein of the S33 class of serine proteases and or has a tertiary structure similar to the prolyl aminopeptidase from Serratia marcescens. It is preferred that amino acids are altered in the protein scaffold at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 47-54, 152-160, 203-212 and 297- 302 in prolyl aminopeptidase from Serratia marcescens, and more preferably at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 50-53, 154-158 and 206-210.
  • the prolyl aminopeptidase from Serratia marcescens or a derivative or homologue thereof is used as the scaffold.
  • the protein scaffold belongs to the S51 class of serine proteases or has at least 70% identity on the amino acid level to a protein of the S51 class of serine proteases and/or has a tertiary structure similar to aspartyl dipeptidase from Escherichia coli.
  • amino acids are altered in the protein scaffold at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 8-16, 38-46, 85-92, 132-140, 159-170 and 205- 211 in aspartyl dipeptidase from Escherichia coli, and more preferably at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 10-14, 87-90, 134-138 and 160-165. It is preferred that the aspartyl dipeptidase from Escherichia coli or a derivative or homologue thereof is used as the scaffold.
  • the protein scaffold belongs to the A2 class of aspartic proteases or has at least 70% identity on the amino acid level to a protein of the A2 class of aspartic proteases and/or has a tertiary structure similar to the protease from human immunodeficiency virus. It is preferred that amino acids are altered in the protein scaffold at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 5-12, 17-23, 27-30, 33-38 and 77-83 in protease from human immunodeficiency virus, and more preferably at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 7-10, 18-21, 34-37 and 79-82.
  • the protease from human immunodeficiency virus preferably HIV-1 protease, or a derivative or homologue thereof is used as the scaffold.
  • the protein scaffold belongs to the A26 class of aspartic proteases or has at least 70% identity on the amino acid level to a protein of the A26 class of aspartic proteases and/or has a tertiary structure similar to the omptin from
  • amino acids are altered in the protein scaffold at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 28-40, 86-98, 150-168, 213-219 and 267-278 in omptin from Escherichia coli, and more preferably at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 33-85 161-168 and 273-277. It is preferred that the omptin from Escherichia coli or a derivative or homologue thereof is used as the scaffold.
  • the protein scaffold belongs to the Cl class of cysteine proteases or has at least 70% identity on the amino acid level to a protein of the Cl class of cysteine proteases and or has a tertiary structure similar to the papain from Carica papaya. It is preferred that amino acids are altered in the protein scaffold at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 17-24, 61-68, 88-95, 135-142, 153-158 and 176-184 in papain from Carica papaya, and more preferably at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 63-66, 136-139 and 177-181.
  • the papain from Carica papaya or a derivative or homologue thereof is used as the scaffold.
  • the protein scaffold belongs to the C2 class of cysteine proteases or has at least 70% identity on the amino acid level to a protein of the C2 class of cysteine proteases and/or has a tertiary structure similar to human calpain.
  • amino acids are altered in the protein scaffold at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 90-103; 160-172, 193-199, 243-260, 286-294 and 316-322 in human calpain-2, and more preferably at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 92-101, 245-250 and 287-291. It is preferred that the human calpain-2 or a derivative or homologue thereof is used as the scaffold.
  • the protein scaffold belongs to the C4 class of cysteine proteases or has at least 70% identity on the amino acid level to a protein of the C4 class of cysteine proteases and or has a tertiary structure similar to Nla protease from tobacco etch virus. It is preferred that amino acids are altered in the protein scaffold at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 23-31, 112-120, 144-150, 168-176 and 205-218 in Nla protease from tobacco etch virus, and more preferably at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 145-149, 169-174 and 212-218.
  • the Nla protease from tobacco etch virus (TEV protease) or a derivative or homologue thereof is used as the scaffold.
  • the protein scaffold belongs to the CIO class of cysteine proteases or has at least 70% identity on the amino acid level to a protein of the CIO class of cysteine proteases and or has a tertiary structure similar to the sfreptopain from Streptococcus pyogenes.
  • amino acids are altered in the protein scaffold at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 81-90, 133-140, 150-164, 191-199, 219-229, 246-256, 306-312 and 330-337 in sfreptopain from Streptococcus pyogenes, and more preferably at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 82-87, 134-138, 250-254 and 331-335. It is preferred that the sfreptopain from Streptococcus pyogenes or a derivative or homologue thereof is used as the scaffold.
  • the protein scaffold belongs to the C19 class of cysteine proteases or has at least 70% identity on the amino acid level to a protein of the C 19 class of cysteine proteases and/or has a tertiary structure similar to human ubiquitin specific protease 7. It is preferred that amino acids are altered in the protein scaffold at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 3-15, 63-70, 80-86, 248-256, 272-283 and 292-304 in human ubiquitin specific protease 7, and more preferably at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 10-15, 251-255, 277-281 and 298-304.
  • the human ubiquitin specific protease 7 or a derivative or homologue thereof is used as the scaffold.
  • the protein scaffold belongs to the C47 class of cysteine proteases or has at least 70% identity on the amino acid level to a protein of the C47 class of cysteine proteases and/or has a tertiary structure similar to the staphopain from Staphylococcus aureus.
  • amino acids are altered in the protein scaffold at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 15-23, 57-66, 108-119, 142-149 and 157- 164 in staphopain from Staphylococcus aureus, and more preferably at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 17-22, 111-117, 143-147 and 159-163. It is preferred that the staphopain from Staphylococcus aureus or a derivative or homologue thereof is used as the scaffold.
  • the protein scaffold belongs to the C48 class of cysteine proteases or has at least 70% identity on the amino acid level to a protein of the C48 class of cysteine proteases and/or has a tertiary structure similar to the UIpl endopeptidase from Saccharomyces cerevisiae.
  • amino acids are altered in the protein scaffold at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 40-51, 108- 115, 132-141, 173-179 and 597-605 in UIpl endopeptidase from Saccharomyces cerevisiae, and more preferably at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 43-49, 110- 113, 133-137 and 175-178. It is preferred that the UIpl endopeptidase from Saccharomyces cerevisiae or a derivative or homologue thereof is used as the scaffold.
  • the protein scaffold belongs to the C56 class of cysteine proteases or has at least 70% identity on the amino acid level to a protein of the C56 class of cysteine proteases and/or has a tertiary structure similar to the Pfpl endopeptidase from Pyrococcus horikoshii.
  • amino acids are altered in the protein scaffold at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 8-16, 40-47, 66-73, 118- 125 and 147-153 in Pfpl endopeptidase from Pyrococcus horikoshii, and more preferably at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 9-14, 68-71 120-123 and 148-151. It is preferred that the Pfpl endopeptidase from Pyrococcus horikoshii or a derivative or homologue thereof is used as the scaffold.
  • the protein scaffold belongs to the M4 class of metallo proteases or has at least 70% identity on the amino acid level to a protein of the M4 class of metallo proteases and/or has a tertiary structure similar to thermolysin from Bacillus thermoproteolyticus.
  • amino acids are altered in the protein scaffold at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 106-118, 125-130, 152-160, 197-204; 210- 213 and 221-229 in thermolysin from Bacillus thermoproteolyticus, and more preferably at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 108-115, 126-129, 199-203 and 223-227. It is preferred that the thermolysin from Bacillus thermoproteolyticus or a derivative or homologue thereof is used as the scaffold.
  • the protein scaffold belongs to the Ml 0 class of metallo proteases or has at least 70% identity on the amino acid level to a protein of the M10 class of metallo proteases and/or has a tertiary structure similar to human collagenase. It is preferred that amino acids are altered in the protein scaffold at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 2-7, 68-79, 85-90, 107-111 and 135-141 in human collagenase, and more preferably at one or more positions from the group of positions that correspond structurally or by amino acid sequence homology to the regions 3-6, 71-78 and 136-140. It is preferred that human collagenase or a derivative or homologue thereof is used as the scaffold.
  • proteases engineered to have new substrate specificity such that they target disease-related molecules.
  • Methods have now been developed to determine the three dimensional structures of proteases that are specificity-programmed to target critical cell surface molecules.
  • Structural data on engineered proteases complexed with target-like peptides provide a framework to understand direct and second shell side chain interactions that determine specificity.
  • the correlation of three dimensional structure and protease activity and specificity are of academic and demonstrated long term clinical interest. The invention moves beyond showing the importance of second shell site alterations in the activity of a protease with altered specificity.
  • the invention provides methods of use and methods for designing and testing disease-specific proteases programmed to target VEGF or VEGFR, which is critical in the etiology of cancer and other diseases. These proteases provide an important new approach to the treatment of cancers, e.g., by impeding tumor growth by blocking tumor angiogenesis, as well as other diseases, including but not limited to macular degeneration, inflammation or diabetes, in which angiogenesis plays a causative or contributive role. The invention also provides methods of use and methods for designing and testing target-specific proteases programmed to target VEGF and VEGFR which are critical for maintaining cancer and other diseases.
  • proteases provide an important new approach to the treatment of cancers, e.g., by impeding tumor growth by blocking tumor angiogenesis, as well as other diseases, including but not limited to macular degeneration, inflammation or diabetes, in which angiogenesis plays a causative or contributive role.
  • the invention also provides methods of use and methods for designing and testing angiogenesis-specific proteases programmed to target proteins critical for modulating apoptosis.
  • These proteases provide an important new approach to the freatment of cancers, e.g., by impeding tumor growth by blocking tumor angiogenesis, as well as other diseases, including but not limited to macular degeneration, inflammation or diabetes, in which angiogenesis plays a causative or contributive role.
  • Methods are provided for specificity determinants in proteases, thereby allowing design of proteases for disabling VEGF or VEGFR.
  • a combination of screening and structure-based mutagenesis is used to design the targeted proteases.
  • Engineering proteases targeted to attack VEGF or VEGFR represents an entirely new sector in the biotechnology industry.
  • Methods are also provided for creating selective proteases as a new therapeutic modality in human disease. Development and proof of concept experiments in animal models of disease provide an understanding of protease substrate selectivity and recognition in this class of enzymes and provide useful information for the dosing and administration of the proteases of the invention for the treatment of human disease.
  • This disclosure provides protease therapeutic agents, methods for their production and reagents useful therewith.
  • the methods use proteases associated with disease to address growing health concerns such as cardiovascular disease, inflammatory disorders and cancer.
  • the invention characterizes the three-dimensional structures of serine proteases with novel extended substrate specificities that are targeted to the vascular endothelial growth factor receptor 2 (VEGF-R2).
  • VEGF-R2 vascular endothelial growth factor receptor 2
  • These proteases were developed using protein engineering and selected using unique and powerful protease profiling technology. Built from wild-type protease scaffolds, they represent a new therapeutic modality in the treatment of cancer. Signaling by vascular endothelial growth factor (VEGF) and its receptors is implicated in pathological angiogenesis and the rapid development of tumor vasculature in cancer.
  • VEGF vascular endothelial growth factor
  • a protease that specifically cleaves and inactivates the signaling of the VEGF/VEGFR-2 complex will attenuate the angiogenic signal and create a pool of soluble receptor that lowers free VEGF levels.
  • Variant proteases have an in vitro specificity that recognizes a critical region of the VEGF receptor, which is, in one embodiment, the Flk-1/KDR stalk, over a six amino acid region. Due to their catalytic nature and smaller size, engineered proteases provide a new therapeutic treatment with advantages over competing targeted binding proteins. The advantages are: better tumor penetration, better target saturation, higher effectiveness, and potentially lower dosing.
  • proteases could cleave and inactivate hundreds to thousands of substrate VEGF receptors, offering substantial therapeutic amplification.
  • some wild-type proteases also cleave VEGFR, and are also used according to the invention.
  • wild-type MT-SPl cleaves VEGFR.
  • Structural data on engineered proteases complexed with target-like peptides provide a framework to understand direct (first) and less direct (second) shell side chain interactions that proscribe specificity. The three dimensional structures of proteases determine its specificity.
  • Residues in the protease scaffold that are important in establishing first and second shell interactions with its substrate are mutated to program the protease to attack various cell surface molecules of interest.
  • the correlation of three dimensional structure and protease activity and specificity provide the following: 1. design and characterization of engineered serine proteases with altered substrate specificity; 2. production and characterization of mutated variants of the macromolecular protease inhibitor ecotin that are mated in specificity to the engineered proteases with substrates and chloromethyl ketone inhibitors; 3. crystallization and determination of the three-dimensional structures for the protease- ecotin complexes; and 4.
  • protease-ecotin structures analyses of the protease-ecotin structures, with subsequent creation of a library of protease and substrate interactions important for selectivity and catalysis. Structures of proteases without substrate analogs are far less helpful than those of proteases with substrate analogs. Substrate analogs are available that bind to many proteases.
  • One powerful agent is ecotin, a serine protease inhibitor found in the periplasm of Escherichia coli. It is a 142-amino acid protein, a novel inhibitor for virtually all serine proteases, regardless of sequence specificity [McGrath, J Biol Chem. 1991 Apr 5;266(10):6620-5.].
  • Ecotin's scissile Met84-Met85 bond lies within a disulfide-bonded protein segment similar to other classes of inhibitors, allowing ecotin to remain bound to the protease even if it is cut.
  • the pan-specificity of inhibition by ecotin derives from formation of a heterotetrameric complex with target proteases involving three types of interface; the dimerization interface, a primary substrate-like interaction, and a smaller secondary interaction between the partner ecotin subunit and the proteases.
  • Ecotin accommodates different shapes of proteases by a balance of the primary and the secondary site. The effect of the secondary binding site on affinity was found to vary inversely with the strength of the interaction at the primary site.
  • the crystal structure of the mutant complex showed surface loops surrounding the active site of thrombin with structural changes to permit inhibitor binding [Wang et al, Biochemistry 2001 Aug 28;40(34): 10038-46].
  • the insertion loops at residues 60 and 148 in thrombin move when the complex forms.
  • the active site of thrombin is filled with eight consecutive amino acids of ecotin and demonstrates thrombin' s preference for specific features: namely, negatively charged Pro-Val-X-Pro-Arg-hydrophobic-positively charged (SEQ ID NO: 19).
  • SEQ ID NO: 19 negatively charged Pro-Val-X-Pro-Arg-hydrophobic-positively charged
  • Chloromethyl ketones are the traditional approach to imaging substrate analogs in proteases and can be used like ecotin to reveal the subsite interaction between a substrate analog and a protease up to the scissile bond. Over sixty structures of these complexes has been determined and, like ecotin, help define the protease specificity mechanism.
  • VEGF-R2 and angiogenic pathology Vascular endothelial growth factor (VEGF) is a cytokine that binds and signals through a specific cell surface receptor (VEGFR) to regulate angiogenesis, the process in which new blood vessels are generated from existing vasculature.
  • VEGFR cell surface receptor
  • Pathological angiogenesis describes the increased vascularization associated with disease and includes events such as the growth of solid tumors [McMahon, Oncologist. 2000;5 Suppl 1:3-10], macular degeneration and diabetes. In cancer, solid tumors require an ever-increasing blood supply for growth and metastasis.
  • VEGF and VEGF-R mRNA in the tumor and surrounding sfromal cells leading to the extension of existing vessels and formation of a new vascular network.
  • abnormal blood vessel growth forms beneath the macula. These vessels leak blood and fluid into the macula damaging photoreceptor cells.
  • VEGF stimulation of capillary growth around the eye leads to disordered vessels which do not function properly.
  • KDR the mouse homolog is Flk-1
  • Flk-1 the mouse homolog is Flk-1
  • KDR-2 the mouse homolog is Flk-1
  • VEGF-R-2/Flk-l/KDR the mouse homolog is Flk-1
  • KDR the mouse homolog is Flk-1
  • VEGF and KDR association has been identified as a key endothelial cell-specific signaling pathway required for pathological angiogenesis [Kim, Nature. 1993 Apr 29; 362 (6423):841-4; Millauer, Nature.
  • Therapies targeting the VEGF receptors and Flk-1/KDR specifically have inhibited pathological angiogenesis and shown reduction of tumor size in multiple mouse models of human and mouse solid tumors [Prewett, Cancer Res. 1999 Oct 15; 59(20):5209-18; Fong, Neoplasia. 1999 Apr; 1(1):31-41. Erratum in: Neoplasia 1999 Jun; 1(2): 183] alone and in combination with cytotoxic therapies [Klement, J Clin Invest. 2000 Apr; 105(8) :R15-24].
  • Studies with small molecule inhibitors and antibodies validate the VEGF receptor family as a potent anti-angiogenesis target but leave room for a more effective therapeutics are still needed.
  • VEGFR is composed of an extracellular region of seven immunoglobin (Ig) -like domains, a transmembrane region, and two cytoplasmic tyrosine kinase domains.
  • Ig immunoglobin
  • the first three Ig-like domains have been shown to regulate ligand binding, while domains 4 through 7 have a role in inhibiting correct dimerization and signaling in the absence of ligand.
  • VEGF-R2 vascular endothelial growth factor-R2
  • Proteases are protein-degrading enzymes that recognize an amino acid or an amino acid substrate sequence within a target protein. Upon recognition of the substrate sequence, proteases catalyze the hydrolysis or cleavage of a peptide bond within a target protein. Such hydrolysis of the target protein can inactivate it, depending on the location of peptide bond within the context of the full-length sequence of the target sequence. The specificity of proteases can be altered through protein engineering. If a protease is engineered to recognize a substrate sequence within a target protein or proteins (i) that would alter the function i.e.
  • the engineered protease has a therapeutic effect via a proteolysis-mediated inactivation event.
  • serine-like proteases e.g. granzyme B and MT-SPl
  • the stalk regions that function to tether protein receptors to the surface of a cell or loop regions are thereby disconnected from the globular domains in a polypeptide chain.
  • the protease cleaves a VEGF or VEGFR which are responsible for modulation of angiogenesis.
  • the cell surface molecule is a VEGFR signaling in tumor angiogenesis
  • cleavage prevents the spread of cancer.
  • cleavage of a cell surface domain from a VEGFR molecule can inactivate its ability to transmit extracellular signals, especially cell proliferation signals. Without angiogenesis to feed the tumor, cancer cells often cannot proliferate.
  • a granzyme B protease of the invention is therefore used to treat cancer.
  • cleavage of VEGFR can be used to modulate angiogenesis in other pathologies, such as macular degeneration, inflammation and diabetes.
  • cleaving a target VEGF or VEGFR protein involved in cell cycle progression inactivates the ability of the protein to allow the cell cycle to go forward. Without the progression of the cell cycle, cancer cells can not proliferate. Therefore, the proteases of the invention which cleave VEGF or VEGFR are used to treat cancer and other cell cycle dependent pathologies. The protease also cleaves soluble proteins that are responsible for tumorigenicity. Cleaving VEGF prevents signaling through the VEGF receptor and decreases angiogenesis, thus decreasing disease in which angiogenesis plays a role, such as cancer, macular degeneration, inflammation and diabetes. Further, VEGF signaling is responsible for the modulation of the cell cycle in certain cell types.
  • the MT- SPl proteases of the invention which cleave VEGF are useful in the treatment of cancer and other cell cycle dependent pathologies.
  • the engineered granzyme B protease is designed to cleave one or more of the target proteins in Table 1, thereby inactivating the activity of the protein.
  • the granzyme B protease can be used to treat a pathology associated with that protein, by inactivating it. Table 1 Protease Targets
  • Granzyme B Multiple sequence alignment of the granzyme B related serine proteases was performed and shown in Table 2. Serine proteases related to granzyme B by distinct active site architecture (no disulfide at amino acids 191 and 220, a truncated 220s loop compared to trypsin, and a cis-Pro at 224) were found. Sequence accession codes from GenBank are reported and the Protein Data Bank identifiers are shown in italics where structures are available. The amino acids with side chains within close contact ( ⁇ 4A) of the substrate are labeled and shown with highlighting. The wild-type granzyme B protease of the invention is provided as SEQ ID NO: 1.
  • Granzyme B belongs to the granzyme subfamily of serine proteases.
  • a ClustalW alignment of mature wild-type human granzyme B (GRAB HUMAN) and other granzyme subfamily members beginning with the canonical N-terminus is provided in Table 2.
  • the granzyme B polypeptide is encoded by the GZMB gene, which resides at chromosome locus 14ql 1.2.
  • the human and rat granzyme B precursor polypeptide of SEQ ID NO:20 and 21 are provided in Table 3.
  • the signal sequence that is cleaved prior to activation is underlined.
  • the GenBank accession number for the human granzyme B protein is M17016 (or 338295) and for rat granzyme B is M34097 (NM_138517).
  • the wild-type mature granzyme B protease of the invention includes residues 21-247 of SEQ IDNOS:20and21.
  • a ClustalW alignment is provided in Table 4, comparing the protease domain of the wild-type human granzyme B polypeptide of SEQ ID NO:l, to the rat granzyme B protease domain of SEQ ID NO:21. Important granzyme B protease domain residues are shown in bold. The bold amino acids are at the specificity determinants, as discussed in more detail below.
  • the wild-type MT-SPl polypeptide of SEQ ID NO:22 is provided in Table 6, and is designated as TADG-15.
  • Table 6 Wild-type MT-SPl polypeptide (SEQ ID NO:22) 1 50 TADG- 15 MGSDRARKGG GGPKDFGAGL KYNSRHEKVN GLEEGVEFLP VNNVKKVEKH 51 100 TADG- -15 GPGRWWLAA VLIGLLLVLL GIGFLVWHLQ YRDVRVQKVF NGYMRITNEN 101 150 TADG- 15 FVDAYENSNS TEFVSLASKV KDALKL YSG VPFLGPYHKE SAVTAFSEGS 151 200 TADG- •15 VIAYYWSEFS IPQHLVEEAE RVMAEERWM LPPRARSLKS FWTSWAFP 201 250
  • a ClustalW alignment is provided in Table 7, comparing the wild-type MT-SPl polypeptide of SEQ ID NO:22, designated as TADG-15, to the MT-SPl protease domain of SEQ ID NO:23.
  • MT-SPl protease domain residues targeted for mutagenesis are shown in bold.
  • the MT-SPl protease domain is composed of a pro-region and a catalytic domain. The catalytically active portion of the sequence begins after the autoactivation site: RQAR followed by the sequence WGG (underlined).
  • a ClustalW alignment is provided in Table 8, comparing the wild-type MT-SPl protease domain of SEQ ID NO:23 with human chymotrypsin (SEQ ID NO:24). MT- SPl protease domain residues targeted for mutagenesis are numbered according to chymotrypsin.
  • Chymotrypsin B VAGEFDQGS-DEENI QVLKIAKVFKNPKFS I TVNNDITLLKLAT PARFSQTVSAVCLPS MTSP_J>rotease_domain FLGLHDQSQRSAPGV QERRLKRIISHPFFN DFTFDYDIALLELEK PAEYSSMVRPICLPD 126 140 141 155 156 170 171 184
  • Chymotrypsin B ADDDFPAGTLCATTG WGKTKYNANKTPDKL QQAALPLLSNAECKK SWGRRITDVMICAG- MTSP_protease_domain ASHVFPAGKAIWVTG WGHTQYGG-TGALIL QKGEIRVINQTTCEN LLPQQITPRMMCVGF 185 198 199 212 213 226 227 240
  • a DNA sequence is provided in Table 9 which encodes the catalytic domain (SEQ ID NO: 19) of wild-type MT-SPl protease domain as contained within the pQE cloning vector.
  • a protease can be re-engineered, including the enzyme substrate sequence specificity, thermostability, pH profile, catalytic efficiency, oxidative stability, and catalytic function.
  • Wild-type protease is used in accordance with the methods of the invention as a scaffold for incorporating various mutations that change its substrate specificity.
  • substrate sequence specificity in serine proteases come from the S1-S4 positions in the active site, where the protease is in contact with the P1-P4 residues of the peptide substrate sequence.
  • the specificity determinants may be generally changed in one pocket without affecting the specificity of the other pockets.
  • a protease with low specificity for a residue at a particular binding site or for a particular sequence is altered in its specificity by making point mutations in the substrate sequence binding pocket.
  • the resulting protease mutein has a greater than 2-fold increase in specificity at a site or for a particular sequence than does wild-type.
  • the resulting protease mutein has a greater than 5-fold increase in specificity at a site or for a particular sequence than does wild-type. In another embodiment, the resulting protease mutein has a greater than 10-fold increase in specificity at a site or for a particular sequence than does wild-type. In another embodiment, the resulting protease mutein has a greater than 100-fold increase in specificity at a site or for a particular sequence than does wild-type. In another embodiment, the resulting protease mutein has an over 1000-fold increase in specificity at a site or for a particular sequence than does wild-type.
  • the specificity is measured by observing how many disparate substrate sequences a mutein protease cleaves at a given activity as compared to the number in the wild-type protease. If the mutein protease cleaves fewer substrate sequences than the wild-type, then the mutein protease has greater specificity than the wild-type. A mutein that has 10 fold higher specificity than a wild-type protease cleaves 10 fold fewer substrate sequences than the wild-type protease. Also contemplated by the invention are libraries of protease scaffolds with various mutations that are generated and screened using methods known in the art and those detailed herein. Libraries are screened to ascertain the substrate sequence specificity of the members.
  • protease scaffolds are tested for specificity by exposing the members to substrate peptide sequences.
  • the library member with the mutations that allow it to cleave the substrate sequence is identified.
  • the protease scaffold library is constructed with enough variety of mutation in the scaffolds that any substrate peptide sequence is cleaved by a member of the library.
  • proteases specific for any target protein can be generated.
  • Particular protease residues that, upon mutation, affect the activity and specificity of scaffold proteases are described here.
  • Serine proteases are mutated and used in aspects of the invention. In one embodiment of the invention, protease muteins with altered specificity are generated by a structure-based design approach.
  • Each protease has a series of amino acids that lines the active site pocket and make direct contact with the substrate. Throughout the chymotrypsin family, the backbone interaction between the substrate and enzyme is conserved, but the side chain interactions vary considerably. The identity of the amino acids that comprise the S1-S4 pockets of the active site determines the substrate specificity of that particular pocket. Grafting the amino acids of one serine protease to another of the same fold may modify the specificity of one to the other. Scaffold residues of serine proteases are identified using chymotrypsin numbering. For example, a mutation at position 99 in the S2 pocket to a smaller amino acid confers a preference for larger hydrophobic residues in the P2 substrate position.
  • proteases are designed with novel substrate specificities towards proteins involved with various diseases.
  • the amino acids of the protease that comprise the S1-S4 pockets are those that have side chains within 4 to 5 angstroms of the substrate.
  • the interactions these amino acids have with the protease substrate are generally called “first shell” interactions because they directly contact the substrate.
  • first shell interactions that ultimately position the first shell amino acids.
  • second shell and “third shell” interactions that ultimately position the first shell amino acids.
  • the invention also contemplates the mutation of those amino actions which undergo second and third shell interactions in order to change the specificity an d rate of reaction of the mutein protease of the invention.
  • Chymotrypsin family members share sequence and structural homology with chymotrypsin. Based on chymotrypsin numbering, the active site residues are Asp 102, His57, and Ser 195.
  • the linear amino acid sequence can be aligned with that of chymotrypsin and numbered according to the ⁇ sheets of chymotrypsin. Insertions and deletions occur in the loops between the beta sheets, but throughout the structural family, the core sheets are conserved.
  • the serine proteases interact with a substrate in a conserved ⁇ sheet manner. Up to 6 conserved hydrogen bonds can occur between the substrate and the enzyme.
  • All serine proteases of the chymotrypsin family have a conserved region at their N-terminus that is necessary for catalytic activity. It is generally IIGG, VVGG or IVGG (SEQ ID NOS:25, 26 and 27, respectively). Where the first amino acid in this quartet is numbered according to the chymotrypsin numbering, it is given the designation of Hel6. This numbering does not reflect the length of the precursor region.
  • Serine protease substrate recognition sites are labeled according to the method of Schecter and Berger (Biochem. Biophys. Res. Commun. 27(1967) 157-162). Labels increase in number from Pl, P2, ... Pn for the substrate amino acids N-terminal to the scissile bond and Pl ', P2', ... Pn' for the substrate amino acids C-terminal to the scissile bond.
  • the corresponding substrate recognition pockets on the enzyme are labeled, Sn ... S2, SI, SI', S2' ... Sn'.
  • P2 interacts with S2, Pl with SI, Pl' with SI', etc.
  • Amino acids in the serine-like protease scaffold are numbered according to their alignment with the serine protease chymotrypsin. See, Blow, D. M. (1976) Ace. Chem. Res. 9, 145-152.
  • the following amino acids in the primary sequence are determinants of specificity: 195, 102, 57 (the catalytic triad); 189, 190,191, 192, and 226 (Pl); 57, and 99 (P2); 192, 217, 218 (P3), the loop between Cysl68 and Cysl80, 215 and 97 to 100 (P4), 41 and 151 (P2').
  • Position 189 in a serine protease is a residue buried at the bottom of the pocket that determines the Pl specificity.
  • the amino acids in the three-dimensional structure that contribute to the substrate selectivity are targeted for mutagenesis.
  • numerous structures of family members have defined the surface residues that contribute to extended substrate specificity (Wang et al, Biochemistry 2001 Aug 28;40(34): 10038-46; Hopfher et al, Structure Fold Des. 1999 Aug 15;7(8): 989-96; Friedrich et al. J Biol Chem. 2002 Jan 18;277(3): 2160-8; Waugh et al, Nat Struct Biol.
  • Structural determinants for granzyme B are listed in Table 10.
  • Table 10 provides a listing of the amino acids in granzyme B determined to be of known, extended specificity. The number underneath the Cysl68-Cysl82 and 60's loop column headings indicate the number of amino acids in the loop between the two amino acids and in the loop. The yes/no designation under the Cysl91-Cys220 column heading indicates whether the disulfide bridge is present in this protease. These regions are variable within the family of chymotrypsin-like serine proteases and represent structural determinants in themselves. Table 10. Structural determinants for granzyme B.
  • Table 11 depicts the potential target cleavage sequences for wild-type and mutein granzyme B. Residues indicated in bold are sequences that are contemplated to be differentially targeted in various individual muteins. Table 11: Substrate specificities for wild-type and mutein granzyme B serine proteases.
  • a mutagenic oligonucleotide primer is synthesized that contains either NNS or NNK-randomization at the desired codon.
  • the primer is annealed to the single stranded DNA template and DNA polymerase is added to synthesize the complementary strain of the template. After ligation, the double stranded DNA template is transformed into E. coli for amplification.
  • single amino acid changes are made using standard, commercially available site-directed mutagenesis kits such as QuikChange (Stratagene).
  • any method commonly known in the art for site specific amino acid mutation of a protease could be used to prepare a set of protease muteins of the invention that can be screened to identify muteins that cleave VEGF, a VEGFR, or another target protein.
  • Granzyme B is a member of the family of chymotrypsin fold serine proteases, and has greater than 50% identity to other members of the granzyme family including granzymes C-G, cathepsin G, and rat mast cell protease II.
  • the protein is a sandwich of two six stranded, anti-parallel ⁇ -barrel domains connected by a short ⁇ -helix.
  • the catalytic triad is composed of Aspl02, His 57 and Ser 195.
  • the surface loops are numbered according to the additions and deletions compared to ⁇ -chymotrypsin and represent the most variable regions of this structural family.
  • the determinants of specificity are defined by the three-dimensional structure of rat granzyme B in complex with ecotin [IEPD (SEQ ID NO: 22)], a macromolecular inhibitor with a substrate-like binding loop (Waugh et al, Nature Struct. Biol). These structural determinants of specificity include Lys 41, Ile99, Argl92, Asn218, Tyr215, Tyrl74, Leul72, Arg226, and Tyrl51, by chymotrypsin numbering.
  • Each modified protease was profiled using a combinatorial substrate library to determine the effect of the mutation on extended specificity. Since the Pl specificity of a protease represents the majority of its specificity, the modifications do not destroy unique specificity of granzyme B towards Pl aspartic acid amino acids but modulate specificity in the extended P2 to P4 sites. For the P3 and P4 subsites, mutations at Tyr 174, Argl 92 and Asn218 did not significantly affect the specificity (See Tables 7 and 8, below). Y174A increases the activity towards Leu at P4, but the rest of the amino acids continue to be poorly selected. R192A and N218A both broaden the specificity at P3.
  • proteases are now preferred nearly 5 times over the average activity of other amino acids at this position.
  • chymotrypsin family of serine proteases more than a dozen proteases have a small residue at this structural site, either an asparagine, serine, threonine, alanine or glycine.
  • two proteases have been profiled using combinatorial substrate libraries, (plasma kallikrein and plasmin), and both show strong preferences towards Phe and Tyr. These two results suggest that any serine protease that is mutated to an Asn, Ser, Thr, Gly or Ala at position 99 will show the same hydrophobic specificity found in plasma kallikrein, plasmin and the I99A granzyme B mutant.
  • P2 specificity determinants may be expanded to the contrasting mutation and substrate preference.
  • Nearly two dozen chymotrypsin-fold serine proteases have an aromatic amino acid at position 99.
  • Four of these proteases have been profiled using combinatorial substrate libraries: human granzyme B, tissue type plasminogen activator, urokinase type plasminogen activator, and membrane type serine protease 1. All but granzyme B have a preference for serine, glycine and alanine amino acids at the substrate P2 position.
  • a mutein from Tables 12A, 12B and 13 activates the substrate by cleavage at the natural granzyme B recognition site.
  • a mutein cleaves granzyme B at a sequence other than the natural site and inactivates it.
  • the muteins described herein are on the rat granzyme B scaffold (RNKP1), but the numbering and residues also apply to the human scaffold. Both human and rat will be made in the expression system of the invention. Table 12A. Granzyme B Mutations
  • granzyme B polypeptides are engineered to selectively cleave and inactivate VEGF receptor 2 (KDR). Wild-type granzyme B protease domain (herein referred to as granzyme B) and mutants thereof are cloned, expressed, purified, and profiled by PSSCL. See, PCT publication WO 01/94332, incorporated by reference herein in its entirety.
  • Wild-type and mutein granzyme B are then assayed for the cleavage of purified VEGF receptor, as further described in the Examples.
  • Granzyme B variants that are able to cleave the purified VEGF receptor are assayed for the cleavage of the receptor on endothelial cells, wherein cleavage results in abrogation of cell proliferation resulting from VEGF signaling. See, e.g. Yilmaz et al, 2003 Biochem. Biophys. Res. Commun. 306(3): 730-736; Gerber et al, 1998 J Biol Chem. 273(46): 30336-43.
  • Promising variants are then tested in animal models angiogenesis and tumor growth, including the mouse micropocket corneal assay and tumor xenographs. See, e.g. Kuo et al., PNAS, 2001, 98:4605-4610.
  • the determinants of specificity selected to be altered in rat granzyme B are shown in Table 15. Mutants of granzyme B were made by QuikChange PCR (Stratagene) according to manufacturer's protocol.
  • Table 15 is a non-limiting listing of a variety of resulting mutant granzyme B polypeptides (muteins), wherein one or more residues in the wild-type granzyme B scaffold are replaced with a residue other than the one at that position in wild-type granzyme B.
  • the granzyme B wild-type residues identified using chymotrypsin numbering, are provided in the left column and the exemplary granzyme B mutant residues are provided in the right column.
  • wild-type Leul71A designates a one amino acid insertion as compared to chymotrypsin.
  • a mutated granzyme B polypeptide may contain a single mutation per polypeptide, or may contain two or more mutated residues in any combination provided in Table 9.
  • an He residue at position 99 is replaced with an Ala residue, wherein the mutein is designated as I99A.
  • other exemplary muteins with a single residue changed include R192A, N218A.
  • Exemplary muteins with two residues changed include I99A N218A, Rl 92A/N218 A and Rl 92E/N218A.
  • Other nonlimiting muteins containing one, two, three, four, five or more replaced residues are provided herein. 2B.
  • Mutagenesis of the MT-SPl Scaffold Protease In order to change the substrate preference of a given subsite (S1-S4) for a given amino acid, the specificity determinants that line the binding pocket are mutated, either individually or in combination.
  • a saturation mutagenesis technique is used in which the residue(s) lining the pocket is mutated to each of the 20 possible amino acids. This can be accomplished using the Kunkle method (In: Current Protocols in Molecular Biology, Ausubel et al. (eds.) John Wiley and Sons, Inc., Media Pa.).
  • a mutagenic oligonucleotide primer is synthesized that contains either NNS or NNK-randomization at the desired codon.
  • the primer is annealed to the single stranded DNA template and DNA polymerase is added to synthesize the complementary strain of the template. After ligation, the double stranded DNA template is transformed into E. coli for amplification.
  • single amino acid changes are made using standard, commercially available site-directed mutagenesis kits such as QuikChange (Stratagene).
  • any method commonly known in the art for site specific amino acid mutation of MT-SPl could be used.
  • MT-SPl is a mosaic protein containing a transmembrane domain, two CUB domains, four LDLR repeats, and a serine protease domain.
  • the protease domain of MT-SPl has been expressed in bacteria or yeast in milligram quantities and purified.
  • Profiling by positional scanning substrate combinatorial libraries (PSSCL) revealed that it has trypsin-like activity, demonstrating a strong preference for basic residues at the Pl position.
  • the extended P2-P4 specificity of MT-SPl is shown in Table 16. Table 16.
  • MT-SPl appears to have a specificity switch, wherein it accepts a positively charged residue in the P4 position or a positively charged residue in the P3 position.
  • the crystal structure of the protease domain of MT-SPl has been solved, providing a structural rationale for its substrate specificity profile.
  • MT-SPl polypeptides are engineered to selectively cleave and inactivate VEGF receptor 2 (KDR) selectively.
  • KDR VEGF receptor 2
  • Wild-type MT-SPl protease domain herein referred to as MT-SPl
  • mutants thereof are cloned, expressed, purified, and profiled by PSSCL.
  • Wild-type and mutant MT-SPl are then assayed for the cleavage of purified VEGF receptor, as further described and illustrated in the Examples below.
  • MT-SPl variants that are able to cleave the purified VEGF receptor are assayed for the cleavage of the receptor on endothelial cells, wherein cleavage results in abrogation of cell proliferation resulting from VEGF signaling. See, e.g. Yilmaz et al, 2003 Biochem. Biophys. Res. Commun. 306(3): 730-736; Gerber et al, 1998 J Biol Chem. 273(46): 30336-43.
  • Promising variants are then tested in animal models angiogenesis and tumor growth, including the mouse micropocket corneal assay and tumor xenografts. See, e.g. Kuo et al, PNAS, 2001, 98:4605-4610.
  • Mutants of MT-SPl were made by QuikChange PCR (Stratagene) according to the manufacturer's protocol.
  • a non-limiting listing of a variety of resulting mutant MT- SPl polypeptides (muteins) is provided in Table 17, and their corresponding CB numbers are provided in Table 18.
  • the MT-SPl wild-type residues, identified using chymotrypsin numbering, are provided in the left column and the MT-SPl mutants are provided in the right column.
  • Asp60b and Arg60c are part of an insertion in MT-SPl not present in chymotrypsin. Therefore all the residues in this loop are assigned to residue 60 when using chymotrypsin numbering.
  • W215Y means that a tryptophan at position 215 of MT-SPl according to the chymotrypsin numbering system is changed to a tyrosine at that position.
  • a mutated MT-SPl polypeptide (“mutein”) may contain a single mutation per polypeptide, or may contain two or more mutated residues per polypeptide in any combination. Exemplary replacements of wild-type residues are provided in Table 18.
  • a Leu residue at position 172 is replaced with an Asp residue, wherein the mutein is designated as L172D.
  • an Asp60b residue is replaced by any one of Ala, Arg, He or Phe.
  • a variant MT-SPl includes at least one of Y146F, L172D, N175D and D217F, and may contain two, three, four or more such residue replacements.
  • the protease is expressed in an active form. In another embodiment, the protease is expressed in an inactive, zymogen form. In one embodiment, the protease is expressed by a heterologously expression system such as an E. coli, Pichiapastoris, S. cerevisiae, or a baculovirus expression system. In a preferred embodiment, the protease is expressed in a mammalian cell culture expression system. Exemplary mammalian cell cultures are derived from rat, mouse, or preferably human cells. The protein can either be expressed in an intracellular environment or excreted (secreted) into the media. The protease can also be expressed in an in vitro expression system.
  • the protease may be engineered to contain a C-terminal 6-His tag for purification on a Nickel column.
  • a cation or anion exchange column can be used in the purification method for the protease.
  • the protease can be stored in a low pH buffer that minimizes its catalytic activity so that it will not degrade itself. Purification can also be accomplished through immunoabsorption, gel filtration, or any other purification method commonly used in the art. The protease can be stored in a low pH buffer that minimizes its catalytic activity so that it will not degrade itself.
  • Essential amino acids in the proteases generated using the methods of the present invention are identified according to procedures known in the art, such as site-directed mutagenesis or saturation mutagenesis of active site residues or disclosed herein.
  • residues that form the S 1 -S4 pockets that have been shown to be important determinants of specificity are mutated to every possible amino acid, either alone or in combination. See, e.g., Legendre, et al, JMB (2000) 296: 87-102.
  • Substrate specificities of the resulting mutants will be determined using the ACC positional scanning libraries and by single substrate kinetic assays. See, e.g., Harris, et al. PNAS, 2000, 97:7754-7759.
  • protease phage display is used to screen the libraries of mutant proteases of the invention for various affinities to specific substrate sequences as described in the art. See, e.g., Legendre et al, JMB, 2000: 296:87-102, and Corey et al, Gene, 1993 Jun 15; 128(1): 129-34.
  • the invention also provides methods for detecting and quantitating an enzymatically active protease of the invention.
  • the method includes: (a) contacting the protease with a library of peptides of the invention in such a manner whereby the fluorogenic moiety is released from the peptide sequence, thereby forming a fluorescent moiety; (b) detecting the fluorescent moiety; and (c) determining the sequence of the peptide sequence, thereby determining the peptide sequence specificity profile of the protease.
  • the method further includes, (d) quantifying the fluorescent moiety, thereby quantifying the protease.
  • the protease can be substantially any protease of interest, but is preferably aspartic protease, cysteine protease, metalloprotease or serine protease.
  • the protease assayed using a method of the invention can be derived from substantially any organism, including, but not limited to, mammals (e.g. humans), birds, reptiles, insects, plants, fungi and the like.
  • the protease is derived from a microorganism, including, but not limited to, bacteria, fungi, yeast, viruses, and protozoa.
  • Example 3 The methods, illustrated in Example 3 can be repeated iteratively or in parallel to characterize a variant protease that has the desired specificity and selectivity at each of the extended binding subsites, P2, P3, and P4.
  • mutations in serine proteases have shown that each of the subsites that form the active site (S1-S4) function independently of one another, such that modification of specificity at one subsite has little influence on specificity at adjacent subsites.
  • engineering substrate specificity and selectivity throughout the extended binding site can be accomplished in a step-wise manner. Mutant proteases that match the desired specificity profiles, as determined by substrate libraries, are then assayed using individual peptide substrates corresponding to the desired cleavage sequence.
  • Variant proteases are also assayed to ascertain that they will cleave the desired sequence when presented in the context of the full-length protein.
  • the activity of the target protein is also assayed to verify that its function has been destroyed by the cleavage event.
  • the cleavage event is monitored by SDS-PAGE after incubating the purified full-length protein with the variant protease.
  • mutant proteases are combined to acquire the specificity of multiple proteases. A mutation at one residue of a scaffold, which produces specificity at one site, is combined in the same protease with another mutation at another site on the scaffold to make a combined specificity protease.
  • the granzyme B scaffold comprises a polypeptide 95% identical to the amino acid sequence of wild-type granzyme B of SEQ ID NO: 1 , and the polypeptide has at least one mutation at one or more of the positions 41, 57, 58, 59, 60, 61, 62, 63, 97, 98, 99, 100, 102, 151, 169, 170, 171, 171A, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 189, 190, 191, 192, 195, 215, 217 or 218, wherein the numbering is for chymotrypsin.
  • the granzyme B mutein is the CB06 construct comprising I99A/N218A/Y174E.
  • the mutein is a CB01 construct comprising I99A N218A (which cleaves both caspase and VEGFR)
  • the mutein is a CB02 comprising I99A N218A/L171E
  • the mutein is a CB05 construct comprising I99A N218A/K172D
  • the mutein is a CB 10 construct comprising I99A/N218A/R192M.
  • a mutein granzyme B embodiment at least one residue is replaced as compared to the granzyme B wild-type polypeptide sequence of SEQ ID NO:20 (human) or SEQ ID NO:21 (rat). Further nonlimiting contemplated granzyme B muteins are provided in Tables 12 and 13.
  • the MT-SPl scaffold comprises a polypeptide 95% identical to the amino acid sequence of wild-type MT-SPl protease domain of SEQ ID NO:23, and the polypeptide has at least one mutation at one or more of the positions 171, 174, 180, 215, 192, 218, 99, 57, 189, 190, 226, 146, 172, 175, 41, 58, 59, 60, 61, 62, 63, 97, 98, 100, 102, 151, 169, 170, 171A, 173, 176, 177, 178, 179, 181, 191, 195 or 224 or 217, wherein the numbering is for chymotrypsin.
  • the mutein is L172D comprising leucine replaced with aspartic acid at position 172.
  • the mutein is Y146F comprising tyrosine replaced with phenylalanine at position 146.
  • the mutein is N175D comprising asparagine replaced with aspartic acid at position 175.
  • the mutein is D217F comprising aspartic acid replaced with phenylalanine at position 217.
  • at least one residue is replaced as compared to the MT-SPl wild-type polypeptide sequence of SEQ ID NO: 1. Further nonlimiting contemplated MT-SPl muteins are provided herein.
  • Proteins targeted for cleavage and inactivation can be identified by the following criteria: 1) the protein is involved in pathology; 2) there is strong evidence the protein is the critical point of intervention for treating the pathology; 3) proteolytic cleavage of the protein will likely destroy its function.
  • VEGF and the VEGFRs are excellent targets for protease-mediated therapies of the invention.
  • Cleavage sites within target proteins are identified by the following criteria: 1) they are located on the exposed surface of the protein; 2) they are located in regions that are devoid of secondary structure (i.e.
  • target protein-assisted catalysis is used to generate proteases specific for a target VEGF or VEGFR protein.
  • a single mutation in the substrate sequence binding site of the protease can alter its specificity and cause it to have a change in substrate sequence specificity.
  • substrate sequence specificity can be altered using a small number of mutations.
  • one of ordinary skill in the art can identify and/or prepare a variety of polypeptides that are substantially homologous to a protease scaffold or allelic variants thereof and retain the proteolysis activity of the wild-type protein scaffold but vary from it in specificity.
  • these polypeptides are based on the scaffold amino acid sequences of granzyme B or MT-SPl.
  • Such polypeptides may optionally include a targeting moiety comprising additional amino acid residues that form an independently folding binding domain.
  • Such domains include, for example, an extracellular ligand-binding domain (e.g., one or more fibronectin type III domains) of a cytokine receptor; immunoglobulin domains; DNA binding domains (see, e.g., He et al, Nature 378:92-96, 1995); affinity tags; and the like.
  • extracellular ligand-binding domain e.g., one or more fibronectin type III domains
  • immunoglobulin domains e.g., one or more fibronectin type III domains
  • DNA binding domains see, e.g., He et al, Nature 378:92-96, 1995
  • affinity tags e.g., affinity tags, and the like.
  • polypeptides may also include additional polypeptide segments as generally disclosed above.
  • the protease muteins and protease libraries of the invention include a polypeptide having an amino acid sequence of one or more of the proteases whose sequence is provided in any one of the scaffolds described herein.
  • the invention also provides a mutant or variant protease any of whose residues may be changed from the corresponding residues shown in any one of the scaffolds described herein, while still encoding a protein that maintains its protease activities and physiological functions, or a functional fragment thereof.
  • the mutations occur in the S1-S4 regions of the protease as detailed herein.
  • a protease variant that preserves protease-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include variants produced by, relative to the wild-type or parent protein sequence, inserting an additional residue or residues between two residues of the parent protein as well as by deleting one or more residues from the parent sequence.
  • Any amino acid substitution, insertion, or deletion is contemplated by the methods, muteins, and mutein libraries of the invention. In favorable circumstances, the substitution is a conservative substitution as described above.
  • One aspect of the invention pertains to isolated proteases, and biologically-active portions thereof, as well as derivatives, fragments, analogs or homologs thereof.
  • proteases of the invention are produced by recombinant DNA techniques.
  • a protease protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques as described above.
  • Biologically-active portions of protease proteins include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences of the full length protease proteins, but with fewer amino acids than the full-length protease proteins, and that exhibit at least one activity of the full length protease protein.
  • biologically-active portions comprise a domain or motif with at least one activity of the protease protein.
  • a biologically-active portion of a protease protein is a polypeptide which is, for example, 10, 25, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 241, 242, 243, 244, 245 or 246 amino acid residues in length, wherein wild-type full length granzyme B is considered to be 247 amino acids in length.
  • a biologically-active portion of a protease protein is a polypeptide which is, for example, 10, 25, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 or more amino acid residues in length, and increasing in amino acid length in whole integers of one (1), up to a length of 855 amino acids, wherein wild-type full length MT-SPl is considered to be 855 amino acids in length (SEQ ID NO:l), and mature is less than 855 aa in length.
  • a "fragment" or a "portion” of a polypeptide contains at least one less amino acid residues than the full length polypeptide.
  • the one or more deleted amino acids may be removed from the N- terminus, the C-terminus, or an internal portion.
  • other biologically-active portions of a protein, from which other regions of the protein have been deleted can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native protease.
  • the protease has an amino acid sequence of one of the scaffolds described herein or one of the mutants of the scaffolds.
  • the protease protein is substantially homologous to one of the scaffolds described herein or one of its muteins, and retains the functional activity of the scaffold protein, yet differs in amino acid sequence due to natural allelic variation or mutagenesis and may differ in specificity as described herein.
  • Representative muteins are disclosed in Tables 12A, 12B, 15, 17 and 18 herein. Determining Homology between Two or More Sequences To determine the percent homology of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence).
  • amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology” is equivalent to amino acid or nucleic acid “identity”).
  • the nucleic acid or amino acid sequence homology may be determined as the degree of identity between two sequences. The homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch, 1970. J Mol Biol 48: 443-453.
  • sequence identity refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C, G, U, or I, in the case of nucleic acids
  • substantially identical denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.
  • Chimeric and Fusion Proteins The invention also provides protease chimeric or fusion proteins.
  • a protease "chimeric protein” or “fusion protein” comprises a protease polypeptide operatively-linked to a non-protease polypeptide.
  • protease polypeptide refers to a polypeptide having an amino acid sequence corresponding to one of the scaffolds described herein or one of the muteins of the scaffolds
  • a non-protease polypeptide refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to one of the scaffolds, e.g., a protein that is different from the scaffolds and that is derived from the same or a different organism.
  • the protease polypeptide can correspond to all or a portion of a parent or scaffold protease protein.
  • a protease fusion protein comprises at least one biologically-active portion of a protease protein. In another embodiment, a protease fusion protein comprises at least two biologically-active portions of a protease protein. In yet another embodiment, a protease fusion protein comprises at least three biologically-active portions of a protease protein.
  • the term "operatively-linked" is intended to indicate that the protease polypeptide and the non-protease polypeptide are fused in-frame with one another. The non-protease polypeptide can be fused to the N-terminus or C-terminus of the protease polypeptide.
  • the fusion protein is a GST-protease fusion protein in which the protease sequences are fused to the N-terminus of the GST (glutathione S-transferase) sequences.
  • GST glutthione S-transferase
  • Such fusion proteins can facilitate the purification of recombinant protease polypeptides.
  • the fusion protein is a Fc fusion in which the protease sequences are fused to the N-terminus of the Fc domain from immunoglobulin G.
  • Such fusion proteins can have better pharmacodynamic properties in vivo.
  • the fusion protein is a protease protein containing a heterologous signal sequence at its N-terminus.
  • protease chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992).
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • a protease-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the protease protein.
  • the invention also pertains to variants of the protease proteins that function as either protease agonists (i.e., mimetics) or as protease antagonists.
  • Variants of the protease protein can be generated by mutagenesis (e.g., discrete point mutation or truncation of the protease protein).
  • An agonist of the protease protein can retain substantially the same, or a subset of, the biological activities of the naturally occurring form of the protease protein.
  • An antagonist of the protease protein can inhibit one or more of the activities of the naturally occurring form of the protease protein by, for example, cleaving the same target protein as the protease protein.
  • treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the protease proteins.
  • VEGF vascular endothelial growth factor
  • VEGFR-2 the inhibition of angiogenic signaling through VEGFR-2 represents an underdeveloped therapeutic area ideal for the development of engineered proteases with novel targeting. Due to their catalytic nature and smaller size, engineered proteases promise a new therapeutic treatment with advantages over competing targeted binding proteins.
  • the expected advantages include, but are not limited to: better tumor penetration, better target saturation, higher effectiveness, and potentially lower dosing. Notably, because they bind, hydrolyze, and release, a single protease could cleave and inactivate hundreds to thousands of substrate VEGF receptors, offering substantial therapeutic amplification.
  • treatment of a pathology comprising administering to a subject in need thereof therapeutically effective amounts of a protease that specifically cleaves and inactivates the signaling of the VEGF/VEGFR-2 complex, such as a in combination with at least one anti-cancer agent.
  • a protease that specifically cleaves and inactivates the signaling of the VEGF/VEGFR-2 complex, such as a in combination with at least one anti-cancer agent.
  • Antiangiogenic therapy has proven successful against both solid cancers and hematological malignancies. See, e.g., Ribatti et al. 2003 J Hematother Stem Cell Res. 12(1), 11-22. Therefore, compositions of the invention provided as antiangiogenic therapy will facilitate the treatment of both hematological and sold tissue malignancies.
  • compositions and methods of treatment provided in the invention may be administered alone or in combination with any other appropriate anti-cancer treatment known to one skilled in the art.
  • the wild-type protease and muteins of the invention can be administered in combination with or in place of AVASTINTM in any therapy where AVASTINTM administration provides therapeutic benefit.
  • the anti-cancer agent is at least one chemotherapeutic agent.
  • the administering of the protease is in combination with at least one radiotherapy.
  • Administration of the combination therapy will attenuate the angiogenic signal and create a pool of soluble receptor that lowers free VEGF levels.
  • a variant granzyme B protease of the invention has an in vitro specificity that matches a critical region of the receptor, the Flk-l/KDR stalk, over a six amino acid region.
  • the serine protease-like mutein polypeptides of the invention may be administered in a composition containing more than one therapeutic agent.
  • the therapeutic agents may be the same or different, and may be, for example, therapeutic radionuclides, drugs, hormones, hormone antagonists, receptor antagonists, enzymes or proenzymes activated by another agent, autocrines, cytokines or any suitable anti-cancer agent known to those skilled in the art.
  • the anti-cancer agent co- administered with the wild-type or mutein protease is AVASTINTM.
  • Toxins also can be used in the methods of the present invention.
  • Other therapeutic agents useful in the present invention include anti-DNA, anti-RNA, radiolabeled oligonucleotides, such as antisense oligonucleotides, anti-protein and anti-chromatin cytotoxic or antimicrobial agents.
  • the antitumor agent may be one of numerous chemotherapy agents such as an alkylating agent, an antimetabolite, a hormonal agent, an antibiotic, an antibody, an anti-cancer biological, Gleevec, colchicine, a vinca alkaloid, L-asparaginase, procarbazine, hydroxyurea, mitotane, nitrosoureas or an imidazole carboxamide.
  • chemotherapy agents such as an alkylating agent, an antimetabolite, a hormonal agent, an antibiotic, an antibody, an anti-cancer biological, Gleevec, colchicine, a vinca alkaloid, L-asparaginase, procarbazine, hydroxyurea, mitotane, nitrosoureas or an imidazole carboxamide.
  • Suitable agents are those agents that promote depolarization of tubulin or prohibit tumor cell proliferation.
  • Chemotherapeutic agents contemplated as within the scope of the invention include, but are not limited to, anti-cancer agents listed in the Orange Book of Approved Drug Products With Therapeutic Equivalence Evaluations, as compiled by the Food and Drug Administration and the U.S. Department of Health and Human Services.
  • the serine protease-like proteases of the invention may also be administered together with radiation therapy treatment. Additional treatments known in the art are contemplated as being within the scope of the invention.
  • the therapeutic agent may be a chemotherapeutic agent.
  • Chemotherapeutic agents are known in the art and include at least the taxanes, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas, triazenes; folic acid analogs, pyrimidine analogs, purine analogs, vinca alkaloids, antibiotics, enzymes, platinum coordination complexes, substituted urea, methyl hydrazine derivatives, adrenocortical suppressants, or antagonists. More specifically, the chemotherapeutic agents may be one or more agents chosen from the non-limiting group of steroids, progestins, estrogens, antiestrogens, or androgens.
  • the chemotherapy agents may be azaribine, bleomycin, bryostatin-1, busulfan, carmustine, chlorambucil, cisplatin, CPT- 11, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, dexamethasone, diethylstilbestrol, doxorubicin, ethinyl estradiol, etoposide, fluorouracil, fluoxymesterone, gemcitabine, hydroxyprogesterone caproate, hydroxyurea, L- asparaginase, leucovorin, lomustine, mechlorethamine, medroprogesterone acetate, megestrol acetate, melphalan, mercaptopurine, methotrexate, methotrexate, mithramycin, mitomycin, mitotane, phenyl butyrate, prednisone, pro
  • chemotherapeutic agent may be before, during or after the administration of the serine protease-like mutein polypeptide.
  • suitable therapeutic agents for use in combination or for co-administration with the proteases of the invention are selected from the group consisting of radioisotope, boron addend, immunomodulator, toxin, photoactive agent or dye, cancer chemotherapeutic drug, antiviral drug, antifungal drug, antibacterial drug, antiprotozoal drug and chemosensitizing agent (See, U.S. Patent Nos. 4,925,648 and 4,932,412).
  • Suitable chemotherapeutic agents are described, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co. 1995), and in Goodman and Gilman's THE PHARMACOLOGICAL BASIS OF THERAPEUTICS (Goodman et al., Eds. Macmillan Publishing Co., New York, 1980 and 2001 editions).
  • Other suitable chemotherapeutic agents such as experimental drugs, are known to those of skill in the art.
  • a suitable therapeutic radioisotope is selected from the group consisting of ⁇ -emitters, ⁇ -emitters, ⁇ -emitters, Auger electron emitters, neutron capturing agents that emit -particles and radioisotopes that decay by electron capture.
  • the radioisotope is selected from the group consisting of 225 Ac, 198 Au, 32 P, 125 1, 131 I, 90 Y, 186 Re, 188 Re, 67 Cu, 177 Lu, 213 Bi, 10 B, and 211 At.
  • the therapeutic agents may comprise different radionuclides, or a drug and a radionuclide.
  • different isotopes that are effective over different distances as a result of their individual energy emissions are used as first and second therapeutic agents in combination with the proteases of the invention.
  • Such agents can be used to achieve more effective treatment of tumors, and are useful in patients presenting with multiple tumors of differing sizes, as in normal clinical circumstances. Few of the available isotopes are useful for treating the very smallest tumor deposits and single cells. In these situations, a drug or toxin may be a more useful therapeutic agent for co-administration with a protease of the invention. Accordingly, in some embodiments of the present invention, isotopes are used in combination with non- isotopic species such as drugs, toxins, and neutron capture agents and co-administered with a protease of the invention. Many drugs and toxins are known which have cytotoxic effects on cells, and can be used in combination with the proteases of the present invention.
  • Drugs that interfere with intracellular protein synthesis can also be used in combination with a protease in the therapeutic the methods of the present invention; such drugs are known to those skilled in the art and include puromycin, cycloheximide, and ribonuclease.
  • the therapeutic methods of the invention may be used for cancer therapy.
  • radioisotopes, drugs, and toxins can be conjugated to antibodies or antibody fragments which specifically bind to markers which are produced by or associated with cancer cells, and that such antibody conjugates can be used to target the radioisotopes, drugs or toxins to tumor sites to enhance their therapeutic efficacy and minimize side effects. Examples of these agents and methods are reviewed in
  • the treatment methods of the invention include those in which a protease of the invention is used in combination with other compounds or techniques for preventing, mitigating or reversing the side effects of certain cytotoxic agents.
  • a protease of the invention is used in combination with other compounds or techniques for preventing, mitigating or reversing the side effects of certain cytotoxic agents.
  • examples of such combinations include, e.g., administration of IL-1 together with an antibody for rapid clearance, as described in e.g., U.S. Pat. No. 4,624,846.
  • Such administration can be performed from 3 to 72 hours after administration of a primary therapeutic treatment with a granzyme B mutein or MT-SPl mutein in combination with a anti-cancer agent (e.g., with a radioisotope, drug or toxin as the cytotoxic component).
  • cancer therapy may involve a combination of more than one tumoricidal agent, e.g., a drug and a radioisotope, or a radioisotope and a Boron- 10 agent for neutron-activated therapy, or a drug and a biological response modifier, or a fusion molecule conjugate and a biological response modifier.
  • the cytokine can be integrated into such a therapeutic regimen to maximize the efficacy of each component thereof.
  • certain antileukemic and antilymphoma antibodies conjugated with radioisotopes that are ⁇ or ⁇ emitters may induce myeloid and other hematopoietic side effects when these agents are not solely directed to the tumor cells. This is observed particularly when the tumor cells are in the circulation and in the blood-forming organs.
  • Concomitant and/or subsequent administration of at least one hematopoietic cytokine e.g., growth factors, such as colony stimulating factors, such as G-CSF and GM-CSF
  • growth factors such as colony stimulating factors, such as G-CSF and GM-CSF
  • radionuclide therapy can be used for the treatment of cancer and other pathological conditions, as described, e.g., in Harbert, "Nuclear Medicine Therapy", New York, Thieme Medical Publishers, 1087, pp. 1-340.
  • a clinician experienced in these procedures will readily be able to adapt the cytokine adjuvant therapy described herein to such procedures to mitigate any hematopoietic side effects thereof.
  • therapy with cytotoxic drugs, co- administered with a protease mutein can be used, e.g., for treatment of cancer, infectious or autoimmune diseases, and for organ rejection therapy.
  • Such treatment is governed by analogous principles to radioisotope therapy with isotopes or radiolabeled antibodies.
  • each therapeutic protease and other therapeutic agents combined with the protease can be effected by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues.
  • Mutein or wild-type proteases and other therapeutic agents can be administered by the same route or by different routes. For example, Mutein or wild-type proteases may be administered by intravenous injection while the other therapeutic agent(s) of the combination may be administered orally.
  • the other therapeutic agent(s) may be administered by intravenous injection.
  • the sequence in which the therapeutic agents are administered is not narrowly critical.
  • Administration of mutein or wild-type proteases also can be accompanied by the administration of the other therapeutic agents as described above in further combination with other biologically active ingredients and non-drug therapies (e.g., surgery or radiation treatment.) or with non-drug therapies alone with mutein or wild-type proteases.
  • the combination therapy further comprises a non-drug treatment
  • the non-drug treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the combination of the therapeutic agents and non-drug treatment is achieved.
  • mutein or wild-type proteases and the other pharmacologically active agent may be administered to a patient simultaneously, sequentially or in combination. If administered sequentially, the time between administrations generally varies from 0.1 to about 48 hours. It will be appreciated that when using mutein or wild-type proteases with other therapeutic agent(s), they may be in the same pharmaceutically acceptable carrier and therefore administered simultaneously. They may be in separate pharmaceutical carriers such as conventional oral dosage forms which are taken simultaneously.
  • a therapy for a angiogenic condition includes mutein or wild-type proteases and AVASTINTM. In one embodiment, this condition is cancer.
  • a therapy for cancer, inflammation, diabetes or macular degeneration includes mutein or wild-type proteases.
  • this therapy further includes another therapeutic as defined above.
  • Advantages attributed to the administration of mutein or wild-type proteases and at least a second agent as part of a specific treatment regimen includes, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutic agents.
  • the co-action of the therapeutic agents is additive.
  • the co-action of the therapeutic agents is synergistic.
  • the co-action of the therapeutic agents improves the therapeutic regimen of one or both of the agents.
  • kits for treating patients having an angiogenic condition such as cancer, comprising a therapeutically effective dose of mutein or wild- type proteases for treating or at least partially alleviating the symptoms of the condition (e.g., AVASTINTM), either in the same or separate packaging, and instructions for its use.
  • an angiogenic condition such as cancer
  • a therapeutically effective dose of mutein or wild- type proteases for treating or at least partially alleviating the symptoms of the condition (e.g., AVASTINTM), either in the same or separate packaging, and instructions for its use.
  • the present invention is suitable for the reduction of cancer symptoms.
  • cancer symptoms include blood in the urine, pain or burning upon urination, frequent urination, cloudy urine, pain in the bone or swelling around the affected site, fractures in bones, weakness, fatigue, weight loss, repeated infections, nausea, vomiting, constipation, problems with urination, weakness or numbness in the legs, bumps and bruises that persist, dizziness, drowsiness, abnormal eye movements or changes in vision, weakness, loss of feeling in arms or legs or difficulties in walking, fits or convulsions, changes in personality, memory or speech, headaches that tend to be worse in the morning and ease during the day, that may be accompanied by nausea or vomiting, a lump or thickening of the breast, discharge from the nipple, change in the skin of the breast, a feeling of heat, or enlarged lymph nodes under the arm, rectal bleeding (red blood in stools or black stools), abdominal cramps, constipation alternating with diarrhea, weight loss, loss of appetite, weakness, pallid
  • These macular degeneration symptoms include blurring of vision, lines forming in vision and gradual or quick loss of vision.
  • the present invention is suitable for the reduction of diabetes symptoms. These diabetes symptoms include loss of vision and blindness.
  • treatment should continue as long as symptoms are suspected or observed
  • the patient status will have improved.
  • Measurement number or frequency of relapses will have decreased, or the time to sustained progression will have increased.
  • the dosage is an important part of the success of the treatment and the health of the patient.
  • the physician has to determine the best dosage for a given patient, according to gender, age, weight, height, pathological state and other parameters.
  • the pharmaceutical compositions of the present invention contain a therapeutically effective amount of mutein or wild-type proteases.
  • the amount of the compound will depend on the patient being treated. The patient's weight, severity of illness, manner of administration and judgment of the prescribing physician should be taken into account in deciding the proper amount.
  • the determination of a therapeutically effective amount of mutein or wild-type proteases or other therapeutic agent is well within the capabilities of one with skill in the art. In some cases, it may be necessary to use dosages outside of the ranges stated in pharmaceutical packaging insert to treat a patient. Those cases will be apparent to the prescribing physician.
  • a physician will also know how and when to interrupt, adjust or terminate treatment in conjunction with a response of a particular patient.
  • Formulation (separately or together) and Administration The compounds of the present invention are administered separately or co- formulated in a suitable co-formulated dosage form.
  • Compounds, including those used in combination therapies are administered to a patient in the form of a pharmaceutically acceptable salt or in a pharmaceutical composition.
  • a compound that is administered in a pharmaceutical composition is mixed with a suitable carrier or excipient such that a therapeutically effective amount is present in the composition.
  • therapeutically effective amount refers to an amount of the compound that is necessary to achieve a desired endpoint (e.g., decreasing symptoms associated with cancer).
  • compositions containing mutein or wild-type proteases and other therapeutic agents can be used to formulate pharmaceutical compositions containing mutein or wild-type proteases and other therapeutic agents.
  • Techniques for formulation and administration may be found in "Remington: The Science and Practice of Pharmacy, Twentieth Edition," Lippincott Williams & Wilkins, Philadelphia, PA. Tablets, capsules, pills, powders, granules, dragees, gels, slurries, ointments, solutions suppositories, injections, inhalants and aerosols are examples of such formulations.
  • the formulations can be administered in either a local or systemic manner or in a depot or sustained release fashion. Administration of the composition can be performed in a variety of ways.
  • compositions and combination therapies of the invention may be administered in combination with a variety of pharmaceutical excipients, including stabilizing agents, carriers and/or encapsulation formulations as described herein.
  • the preparation of pharmaceutical or pharmacological compositions will be known to those of skill in the art in light of the present disclosure.
  • such compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection; as tablets or other solids for oral administration; as time release capsules; or in any other form currently used, including creams, lotions, mouthwashes, inhalants and the like.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by the FDA.
  • Administration of compounds alone or in combination therapies may be, e.g., subcutaneous, intramuscular or intravenous injection, or any other suitable route of administration.
  • a particularly convenient frequency for the administration of the compounds of the invention is once a day.
  • therapeutics will be administered in a manner compatible with the dosage formulation, and in such amount as is pharmacologically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the injectable solutions described, but drug release capsules and the like can also be employed.
  • the quantity of active ingredient and volume of composition to be administered depends on the host animal to be treated. Precise amounts of active compound required for administration depend on the judgment of the practitioner and are peculiar to each individual. A minimal volume of a composition required to disperse the active compounds is typically utilized.
  • a carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Suitable preservatives for use in solution include benzalkonium chloride, benzethonium chloride, chlorobutanol, thimerosal and the like.
  • Suitable buffers include boric acid, sodium and potassium bicarbonate, sodium and potassium borates, sodium and potassium carbonate, sodium acetate, sodium biphosphate and the like, in amounts sufficient to maintain the pH at between about pH 6 and pH 8, and preferably, between about pH 7 and pH 7.5.
  • Suitable tonicity agents are dextran 40, dextran 70, dextrose, glycerin, potassium chloride, propylene glycol, sodium chloride, and the like, such that the sodium chloride equivalent of the ophthalmic solution is in the range 0.9 plus or minus 0.2%.
  • Suitable antioxidants and stabilizers include sodium bisulfite, sodium metabisulfite, sodium thiosulfite, thiourea and the like.
  • Suitable wetting and clarifying agents include polysorbate 80, polysorbate 20, poloxamer 282 and tyloxapol.
  • Suitable viscosity-increasing agents include dextran 40, dextran 70, gelatin, glycerin, hydroxyethylcellulose, hydroxmethylpropylcellulose, lanolin, methylcellulose, petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxymethylcellulose and the like.
  • the compounds and combination therapies of the invention can be formulated by dissolving, suspending or emulsifying in an aqueous or nonaqueous solvent.
  • Vegetable e.g., sesame oil, peanut oil
  • Aqueous solutions such as Hank's solution, Ringer's solution or physiological saline buffer can also be used. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Solutions of active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions
  • the preferred methods of preparation are vacuum-drying and freeze- drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the preparation of more, or highly, concentrated solutions for subcutaneous or intramuscular injection is also contemplated.
  • the use of DMSO as solvent is preferred as this will result in extremely rapid penetration, delivering high concentrations of the active compound(s) or agent(s) to a small area.
  • one or both active ingredients of the combination therapy is given orally, it can be formulated through combination with pharmaceutically acceptable carriers that are well known in the art.
  • the carriers enable the compound to be formulated, for example, as a tablet, pill, capsule, solution, suspension, sustained release formulation; powder, liquid or gel for oral ingestion by the patient.
  • Oral use formulations can be obtained in a variety of ways, including mixing the compound with a solid excipient, optionally grinding the resulting mixture, adding suitable auxiliaries and processing the granule mixture.
  • excipients that can be used in an oral formulation: sugars such as lactose, sucrose, mannitol or sorbitol; cellulose preparations such as maize starch, wheat starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidone (PVP).
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like.
  • oral pharmaceutical compositions will comprise an inert diluent or assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
  • the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations should contain at least 0.1% of active compound.
  • the percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 75% of the weight of the unit, or preferably between 25-60%.
  • the amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained.
  • the tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, comstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as com starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring.
  • a binder as gum tragacanth, acacia, comstarch, or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as com starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stea
  • the dosage unit form When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both.
  • a syrup of elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabensas preservatives, a dye and flavoring, such as cherry or orange flavor. Additional formulations suitable for other modes of administration include suppositories.
  • binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1%- 2%.
  • the subject treated by the methods of the invention is a mammal, more preferably a human.
  • the following properties or applications of these methods will essentially be described for humans although they may also be applied to non-human mammals, e.g., apes, monkeys, dogs, mice, etc.
  • the invention therefore can also be used in a veterinarian context.
  • the following examples are nonlimiting and meant only to illustrate various aspects of the invention. EXAMPLES
  • Example 1 Methods of cloning and characterizing engineered granzyme B protease with altered substrate specificity based on well understood starting scaffolds.
  • the serine protease granzyme B has been chosen as the scaffold protease for mutagenesis towards specific proteolysis of the VEGF and VEGFR in part because it has been well characterized with biochemical and structural techniques [Harris et al, J Biol Chem, Vol. 273, Issue 42, 27364-27373, October 16, 1998; Waugh, S. M., Harris, J. L., Fletterick, R., and Craik, C. S. (2000) Nature Structural Biology 7(9), 762-765.].
  • Granzyme B is a serine protease involved in cytotoxic lymphocyte induced cell death of tumor or virally infected cells.
  • PSSCL results for rat granzyme B.
  • the activity in each subsite well is normalized to 100%):
  • the three dimensional structure of rat granzyme B shown as a ribbon and as a surface (FIGS. 5E-5F).
  • the N-terminal side of the ecotin inhibitor's substrate-like binding loop is shown in medium grey, and the C-terminal side of the substrate is modeled in light grey.
  • the ribbon structure of granzyme B mutein CB06 is provided in FIG. 6.
  • a mutated granzyme B polypeptide may contain a single mutation per polypeptide, or may contain two or more mutated residues in any combination provided in Tables 12, 13 and 14.
  • Wild-type and mutant granzyme B are cloned into the pPICZalphaA yeast expression vector (Invitrogen) flush against the alpha factor secretion signal, and transformed into X33 cells. Cells are grown in 250 mL cultures to an OD of 0.6 and expression of the secreted, active protease is induced by adding methanol to a final concentration of 0.5%.
  • the yeast are pelleted by centrifugation and the supernatant is loaded onto a SP-Sepharose Fast Flow resin (Amersham).
  • the column is washed with three volumes each of 50 mM MES pH 6.0, 50 mM NaCl and 200 mM NaCal.
  • the protease is then eluted with three (3) column volumes of 50 mM MES ⁇ H6.0, 1 M NaCl, and the column is washed with 50 mM MES pH 6.0, 2M NaCl.
  • the protease is then concentrated to a volume of approximately 1 mL and exchanged into a storage buffer consisting of 50 mM MES pH 6.0, 100 mM NaCl. Result. Multimilligram quantities are obtained using a yeast expression system.
  • protease is secreted in its mature, catalytically active form and purified with a one-column purification procedure.
  • Fmoc- amino acids (4.8 mmol, 10 equiv/well; Fmoc-amino acid, mol %: Fmoc-Ala-OH, 3.4; Fmoc-Arg(Pbf)-OH, 6.5; Fmoc-Asn(Trt)-OH, 5.3; Fmoc-Asp(O-t-Bu)-OH, 3.5; Fmoc- Glu(O-t-Bu)-OH, 3.6; Fmoc-Gln(Trt)-OH, 5.3; Fmoc-Gly-OH, 2.9; Fmoc-His(Trt)-OH, 3.5; Fmoc-Ile-OH, 17.4; Fmoc-Leu-OH, 4.9; Fmoc-Lys(Boc)-OH, 6.2; Fmoc-Nle-OH, 3.8
  • the solution (0.5 mL) was added to each of the wells.
  • the reaction block was agitated for 3 h, filtered, and washed with DMF (3 x 0.5 mL).
  • the randomized P3 and P4 positions were incorporated in the same manner.
  • the Fmoc of the P4 amino acid was removed and the resin was washed with DMF (3 x 0.5 mL), and treated with 0.5 mL of a capping solution of AcOH (150 ⁇ L, 2.5 mmol), HOBt (340 mg, 2.5 mmol) and DICI (390 ⁇ L, 2.5 mmol) in DMF (10 mL).
  • the resin was washed with DMF (3 x 0.5 mL), CH 2 C1 2 (3x 0.5 mL), and treated with a solution of 95:2.5:2.5
  • 7-Fmoc-aminocoumarin-4-acetic acid was prepared by treating 7- aminocoumarin-4-acetic acid with Fmoc-Cl.
  • 7-Aminocoumarin-4-acetic acid (10.0 g, 45.6 mmol) and H 2 O (228 ml) were mixed.
  • NaHCO 3 (3.92 g, 45.6 mmol) was added in small portions followed by the addition of acetone (228 ml).
  • the solution was cooled with an ice bath, and Fmoc-Cl (10.7 g, 41.5 mmol) was added with stirring over the course of 1 h. The ice bath was removed and the solution was stirred overnight.
  • ACC-resin was prepared by condensation of Rink Amide AM resin with 7-Fmoc-aminocoumarin-4- acetic acid.
  • Rink Amide AM resin (21 g, 17 mmol) was solvated with DMF (200 ml). The mixture was agitated for 30 min and filtered with a filter cannula, whereupon 20% piperidine in DMF (200 ml) was added. After agitation for 25 min, the resin was filtered and washed with DMF (3 times, 200 ml each).
  • the solution (0.5 ml) was added to each of the wells.
  • the reaction block was agitated for 3 h, filtered, and washed with DMF (three times with 0.5 ml).
  • the randomized P3 and P4 positions were incorporated in the same manner.
  • the Fmoc of the P4 amino acid was removed and the resin was washed with DMF (three times with 0.5 ml) and treated with 0.5 ml of a capping solution of AcOH (150 ⁇ l, 2.5 mmol), HOBt (340 mg, 2.5 mmol), and DICI (390 ⁇ l, 2.5 mmol) in DMF (10 ml).
  • the resin was washed with DMF (three times with 0.5 ml) and CH 2 C1 2 (three times with 0.5 ml), and treated with a solution of 95:2.5:2.5 TFA/TIS/H 2 O.
  • the reaction block was opened and placed on a 96-deep-well titer plate and the wells were washed with additional cleavage solution (twice with 0.5 ml).
  • the collection plate was concentrated, and the material in the substrate-containing wells was diluted with EtOH (0.5 ml) and concentrated twice.
  • the contents of the individual wells were lyophilized from CH 3 CN/H 2 O mixtures.
  • the total amount of substrate in each well was conservatively estimated to be 0.0063 mmol (50%) on the basis of yields of single substrates.
  • Pl -substituted ACC-resin Screening Methods Using Both Libraries
  • Multigram quantities of Pl -substituted ACC-resin can be synthesized by the methods described. Fmoc-amino acid-substituted ACC resin was placed in 57 wells of a 96-well reaction block: sub-libraries were denoted by the second fixed position (P4, P3, P2) of 19 amino acids (cysteine was omitted and norleucine was substituted for methionine).
  • Variant proteases were diluted in protease activity buffer (50 mM Na Hepes, pH 8.0, 100 mM NaCl, 0.01% Tween-20) to concentrations between 50 nM and 1 ⁇ M. Initial activity against Ac-QGR-AMC was used to adjust the variant protease concentration to one approximately equal to 50 nM wild-type protease. Enzymatic activity in the Pl-Arg library was assayed for one hour at 30 °C on a Spectra- Max Delta flourimeter (Molecular Devices). Excitation and emission were measured at 380 nm and 460 nm, respectively.
  • Hydrolysis reactions were initiated by the addition of enzyme (0.02 nM-100 nM) and monitored fluorimetrically with a Perkin Elmer LS50B Luminescence Spectrometer, with excitation at 380 nm and emission at 450 nm or 460 nm.
  • Assays of the serine proteases were performed at 25 °C. in a buffer containing 50 mM Tris, pH 8.0, 100 mM NaCl, 0.5 mM CaCl 2 , 0.01% Tween-20, and 1% DMSO (from substrates).
  • Assay of the cysteine proteases, papain and cruzain was performed at 25 °C.
  • the P2, P3, and P4 positions consist of an equimolar mixture of all amino acids for a total of 6,859 substrate sequences per well.
  • Several serine and cysteine proteases were profiled to test the applicability of this library for the identification of the optimal Pl amino acid.
  • Chymotrypsin showed the expected specificity for large hydrophobic amino acids. Trypsin and thrombin showed preference for Pl -basic amino acids (Arg>Lys). Plasmin also showed a preference for basic amino acids (Lys>Arg).
  • Granzyme B the only known mammalian serine protease to have Pl- Asp specificity, showed a distinct preference for aspartic acid over all other amino acids, including the other acidic amino acid, Glu.
  • the Pl -profile for human neutrophil elastase has the canonical preference for alanine and valine.
  • the cysteine proteases, papain and cruzain showed the broad Pl -substrate sequence specificity that is known for these enzymes, although there is a modest preference for arginine.
  • the MT-SPl wild-type protease preferred Arg or Lys.
  • C. Profiling Proteases with the Pi-Constant Library A PI- constant tetrapeptide library is created as disclosed above.
  • the Pl -constant library consists of 20 wells in which only the Pl -position is systematically held constant as all amino acids, excluding cysteine and including norleucine.
  • the P2, P3, and P4 positions consist of an equimolar mixture of all amino acids for a total of 6,859 substrate sequences per well.
  • Several serine and cysteine proteases were profiled to test the applicability of this library for the identification of the optimal Pl amino acid.
  • MT-SPl prefers the amino acids Arg and Lys at Pl.
  • the Pi-Asp fixed PSSCL library is resuspended in DMSO and arrayed in opaque black 96-well plates at a concentration of 5-10 nanomoles per well.
  • Variant proteases are diluted into 50 mM Tris pH 8, 50 mM NaCl, and 0.01% Tween 20 (granzyme B activation buffer) at a concentration of 10-100 nM.
  • One hundred microliters of the protease solution is added to each well and fluorescence of the ACC leaving group is measured by excitation at 380 nm and emission at 460 nm using a Spectramax fluorescent plate reader (Molecular Devices).
  • the specificity of variant proteases at each of the P4-P2 extended subsites is determined by the rate of increase in the fluorescence over time of each of the arrayed amino acids in the P4-P2 PSSC libraries.
  • the Pl residue is held constant as Asp. Screening by PSSCL confirms that wild-type granzyme B has a preference for I/V at P4, E at P3, P/T at P2 and D at Pl (FIG. 7), in agreement with published data.
  • VEGF-R2/KDR polypeptide sequence of VEGF receptor 2
  • Table 19 Sequences that closely match the P4-P1 native substrate specificity of granzyme B or the novel specificities of CBOl and CB06 are shown in bold, and include VLKD, LVED and WFKD. Table 19.
  • Granzyme B was engineered to obtain muteins that exclusively cleave Flk-l/KDR.
  • I99AN218A CB02 (I99A/N218AL171E), CB05(I99A/N218AK172D), CB06 (I99AN218A/Y174E), CB07 (I99A/N218A/Y174D) and CB10 (I99AN218AR192M) are first generation results of this work with novel extended specificity with modified substrate selectivity at the P2 through P4 positions.
  • These variants and the wild-type granzyme B proteases were evaluated for their ability to catalyze in vitro cleavage of a soluble Flkl-Fc fusion protein consisting of the entire Flkl ectodomain fused to an IgG2a antibody Fc fragment.
  • Wild-type granzyme B and the listed muteins cleave the VEGFR-Fc receptor (FIG. 10). These preliminary in vitro experiments confirm that variant proteases will cut the receptor at specific sequences and that the identified target sequences are accessible to proteolysis.
  • the cleavage site of CBOl and wild-type granzyme B were identified from the gel by the Edman N-terminal sequencing method.
  • Flkl-Fc is cleaved by wild-type and granzyme B muteins at the sequence LVED/SGID Example 6.
  • Cleavage by variant proteases yields cleavage products with apparent molecular weights of ⁇ 80 kDa and 30 kDa; analysis of potential cleavage sites in VEGFR2 suggests that variants target the stalk (membrane proximal) region of VEGFR2. All mutants cleave full-length VEGFR2, some at a reduced rate compared to the wild-type, while others cleave the receptor with higher efficiency than wild-type. None of the protease variants (wild-type or mutant) cleave the Fc domain.
  • Example 7 Granzyme B is secreted in an active form from mammalian cells.
  • HEK293T Human embryonic kidney cells
  • DNA constructs encoding the granzyme B protease domain fused to Fc with a N-terminal IgK leader sequence were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. After 40 hours, 100 ⁇ L of the medial were collected from each well, to which was added a fluorogeneic granzyme B substrate (Ac-IETD-AMC, BaChem) to a final concentration of 200 ⁇ M.
  • Granzyme B activity was detected using a Gemini XS fluorescence plate reader (Molecular Devices) with an excitation wavelength of 380 nm and an emission wavelength of 450 nm.
  • both granzyme B and grB-Fc are secreted in active forms from HEK293T cells, as evidenced by granzyme B- specific cleavage of a substrate. There is no activity in the control wells where there was no DNA added, suggesting that HEK293T cells normally do not make and secrete granzyme B.
  • the cells were grown in media containing 10% fetal calf serum, the fact that granzyme B was found to be active in this media suggests that macromolecular inhibitors present in serum are not effective at inhibiting granzyme B.
  • Rat granzyme B was cloned into pAdd or pAdd-Fc vectors for adenoviral delivery in mice according to Kuo et al., PNAS, 2001, 98:4605-4610.
  • C57BL/6 mice receive 5 x 10 8 pfu i.v. of protease-encoding adenoviruses or the control adenovims Ad Fc 2 days before assay.
  • Mice are anesthetized with avertin i.p. and the eye was treated with topical proparacaine-HCl (Allergan, Irvine, CA).
  • Hydron/sucralfate pellets containing VEGF-A 165 are implanted into a comeal micropocket at 1 mm from the limbus of both eyes under an operating microscope (Zeiss) followed by intrastomal linear keratotomy by using a microknife (Medtroni Xomed, Jacksonville, FL).
  • a comeal micropocket is dissected toward the limbus with a von Graefe knife #3 (2 30 mm), followed by pellet implantation and application of topical erythromycin.
  • neovascularization is quantitated by using a slit lamp biomicroscope and the formula 2 ⁇ x (vessel length 10) x (clock hours).
  • mice were bled at varying time points over the course of 3 weeks. Two microliters of semm was separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with a polyclonal antibody specific to granzyme B. Results. Injection of adenovims encoding granzyme B into mice resulted in robust expression and secretion of the protease into semm, with maximal expression occurring on day 2 and extending until day 5 (Fig. 13 A).
  • Murine Lewis lung carcinoma (LLC) cells are passaged on the dorsal midline of C57BL/6 mice or in DMEM/10% FCS/penicillin streptomycin(PNS)/L-glutamine.
  • T241 murine fibrosarcoma is grown in DMEM/10% FCS/PNS/L-glutamine and human pancreatic BxPc3 adenocarcinoma in RPMI medium 1640/10% FCS/PNS.
  • Tumor cells (10 6 ) are injected s.c.
  • Tumor size in mm is calculated by caliper measurements over a 10- to 14-day period by using the formula 0.52 length (mm) width (mm), using width as the smaller dimension. See, e.g., Kuo et al., PNAS, 2001, 98:4605-4610.
  • the invention provides variants of the macromolecular protease inhibitor ecotin that are mated to the engineered proteases for defining the active site.
  • Ecotin is a powerful tool for defining the active sites of serine protease due to the extended substrate-like interaction that it makes with the protease. It takes a molecular impression of the active site revealing features of substrate preference.
  • the 80's loop of ecotin binds to the protease in an extended beta sheet conformation.
  • Site directed mutagenesis is used to mutate amino acids 81 through 86 to match the substrate specificity of interest.
  • an ecotin variant is made that matches the extended specificity in the Pl through P4 positions (residues 81 through 84 of ecotin).
  • the inhibition constant (Kj) of the protease/inhibitor complex is then measured using protocols established in the art and derived from tight binding equations.
  • Each protease is measured with a specific fluorogenic tetrapeptide substrate that matches the proteases specificity of the variant mutein, and with an ecotin that matches the specificity and the target sequence.
  • some ecotins with altered reactive sites loops did not form tight complexes with the substrate and instead were proteolyzed. If this occurs, a tetrapeptide chloromethyl ketone inhibitors is provided with matching specificity for the crystallography.
  • Example 11 Crystallization and determination of 3-dimensional complex structures.
  • the wild-type rat granzyme B construct was prepared as described previously (Harris et al, JBC, 1998, (273):27364-27373).
  • the following point mutations were introduced into the pPICZ ⁇ A plasmid: N218A, N218T, N218V, I99A, I99F, I99R, Yl 74A, Yl 74V.
  • Each mutation was confirmed by sequencing with primers to the 5'AOX and 3'AOX regions, followed by transformation into X33 cells and selection with Zeocin (Invitrogen, La Jolla CA).
  • a polymerase chain reaction was made containing the wild-type double stranded DNA, the two primers overlapping the mutation, a reaction buffer, dNTP's and the DNA polymerase. After 30 rounds of annealing and amplification, the reaction was stopped. The enzyme Dpnl was added to digest the wild-type DNA containing a modified base pair, and the resulting nicked DNA strand is transformed into bacteria. A selection against Zeocin ensures only positive clones with grow. The mutation was confirmed by sequencing the granzyme B gene. The same protocol was used to make the remaining granzyme B mutants, with appropriate changes in the mutagenic primers. The DNA containing the variant granzyme B proteases was transformed into
  • Pichia pastoris X33 cells by the published protocol (Invitrogen) and the positive transformants were selected with Zeocin.
  • To purify the variant protease the culture was centrifuged and the supernatant collected. Gravity based loading flowed the supernatant over a SP-Sepharose Fast Flow cation exchange column. The column was washed with 50 mM Mes, pH 6.0, 100 mM NaCl, and more stringently with 50 mM MES, pH 6.0, 250 mM NaCl.
  • the protein was eluted with 50 mM MES, pH 6.0, 1 M NaCl and the column washed with 50 mM MES, pH 6.0, 2M NaCl and 0.5 M NaOH.
  • the resulting protease was ⁇ 90% pure.
  • the final protease was exchanged and concentrated into 50mM MES, pH 6.0, 100 mM NaCl for storage at 4 °C.
  • Example 14 Screening for cleavage of individual substrates Mutant proteases that match the desired specificity profiles, i.e. cleave a sequence present in VEGFR as determined by substrate libraries, are assayed using individual peptide subsfrates co ⁇ esponding to the desired cleavage sequence. Individual kinetic measurements are performed using a Spectra-Max Delta fluorimeter (Molecular Devices). Each protease is diluted to between 50 nM and 1 ⁇ M in assay buffer. All ACC substrates are diluted with MeSO to between 5 and 500 ⁇ M, while AMC substrates are diluted to between 20 and 2000 ⁇ M. Each assay contain less than 5% MeSO.
  • Enzymatic activity is monitored every 15 seconds at excitation and emission wavelengths of 380 nm and 460 nm, respectively, for a total of 10 minutes. All assays are performed in 1%> DMSO. Example 15. Screening for cleavage of full-length proteins
  • Variant proteases are assayed to ascertain that they will cleave the desired sequence when presented in the context of the full-length protein, and the activity of the target protein is assayed to verify that its function has been destroyed by the cleavage event.
  • the cleavage event is monitored by SDS-PAGE after incubating the purified full-length protein with the variant protease.
  • the protein is visualized using standard Coomasie blue staining, by autoradiography using radio labeled protein, or by Western blot using the appropriate antibody.
  • the target protein is a cell surface receptor, cells expressing the target protein are exposed to the variant protease.
  • the cleavage event is monitored by lysing the cells and then separating the proteins by SDS-PAGE, followed by visualization by Western blot. Alternatively, the soluble receptor released by proteolysis is quantified by ELISA.
  • Cleavage Of VEGFR 125 I- VEGFR (40,000 cpm) is incubated with varying concentrations of protease, samples are boiled in SDS-PAGE sample buffer and examined on a 12% polyacrylamide gel. The gels are dried and exposed to x-ray film (Kodak) at -70 °C.
  • VEGFR Binding Assay 125 I- VEGFR or PMN are incubated with varying concentrations of proteases as above.
  • Cytotoxic lymphocytes are the major immune system defense against virally and tumor infected cells. They efficiently initiate apoptosis in the target cell by the directional release of cytotoxic granules.
  • cytotoxic granules Within these granules is a family of serine proteases known as the granzymes along with the pore forming protein perform and serglycan.
  • the granzymes are a family of trypsin-like serine proteases and thus are composed of two ⁇ ba ⁇ el domains that bind a peptide substrate in an extended ⁇ strand interaction composed of at least four backbone hydrogen bonds.
  • pre-pro-proteases where the pre region targets them to the granules, and activation occurs through the proteolytic processing of the pro region by dipeptidyl protease 1.
  • dipeptidyl protease 1 Of particular interest in this family is granzyme B, a well characterized serine protease with specificity for aspartic acid that is unique among serine proteases. Native granzyme B initiates the apoptotic cascade by cleaving caspases 3 and 7 and contributes to cytoplasmic and nuclear hallmarks of cell death by proteolyzing a specific set of intercellular substrates.
  • the strict requirement by granzyme B for an aspartic acid residue at the primary, or Pl position is unique among mammalian enzymes as is the requirement for a specific extended substrate containing Ile/Val at the P4 position, Glu/Gln/Met at the P3 position, any amino acid at the P2 position, non-charged amino acids at the Pl ' position and Ser/Ala/Gly at the P2' position. It is located in a tightly linked gene cluster on chromosome 14 of the rat, mouse and human species along with chymase, cathepsin G, rat mast cell protease and granzymes C through H.
  • This subfamily of serine proteases are homologous, sharing >46% identity and >56% similarity, yet they have at least five identified or predicted primary specificities and unique extended subsfrates.
  • the na ⁇ ow substrate specificity of such regulatory serine proteases is thought to occur through a constellation of amino acids identified by x-ray crystallography surrounding the active site. The specific interactions that determine na ⁇ ow extended substrate specificity occur between substrate and protease side chains on the highly variable loops between the ⁇ strands.
  • trypsin-fold serine proteases the primary site amino acids 189, 191, 216, and 226 and the loops containing them have been investigated through protein engineering for their roles in Pl specificity in other serine proteases.
  • the high sequence identity of the granzyme B-like subfamily na ⁇ ows the number of possible extended specificity determinants, and suggests that mutagenic changes will introduce radical alterations in extended specificity.
  • the coincidence of crystal structures of granzyme B, chymase, rat mast cell protease and cathepsin G and combinatorial assays for determining substrate specificity provide a powerful basis for understanding extended subsfrate specificity using site-directed mutagenesis. This study seeks to understand the contribution of amino acids identified by the crystal stmcture of rat granzyme B to its extended specificity, and the role of mutations in the selective modification of its specificity.
  • the Asp-ACC conjugated resin was a gift from the lab of John Ellman (UC Berkeley).
  • the Pl-Asp-AMC library was a gift of Nancy Thornberry (Merck Laboratories, Rahway, NJ).
  • the complete diverse ACC tetrapeptide substrate library was a gift of Youngchool Choe (Craik Lab). Alignment of granzyme B sub-family serine proteases Rat granzyme B was used as a query to find related granzyme and granule proteases from the NCBI database (National Library of Medicine, National Institutes of Health, Bethesda, MD) using PSI-BLAST and HSSP.
  • the sequences were aligned using ClustalW 8.1.
  • the amino acid sequence of rat granzyme B is found under the NCBI Accession #NP_612526 or the Swiss-Prot # 18291.
  • the x-ray crystallographic stmcture of rat granzyme B complexed with ecotin [81-84 IEPD] was used to determine amino acids within 4 A of the substrate amino acids. Amino acids were also evaluated that contact small molecule inhibitors in the human granzyme B, chymase, cathepsin G and rat mast cell protease II structures.
  • rat granzyme B PDB # 1FI8
  • human granzyme B PDB # 1FQ3 and 1IAU
  • chymase PDB # 1PJP and 1KLT
  • cathepsin G PDB # 1AU8 and 1CGH
  • rat mast cell protease 11 PDB # 3RP2.
  • Variants of rat granzyme B The wild-type, Rl 92A, and Rl 92E rat granzyme B constructs were prepared as described above. Additional variants of wild-type rat granzyme B were constructed using the QuikChange protocol (Stratagene, La Jolla, CA).
  • N218A GGC ATC GTC TCC TAT GGA CAA GCT GAT GGT TCA ACT CCA CGG GCA (SEQ ID NO:37), I99A: CCA GCG TATAAT TCTAAGACAGCC TCC AAT GACATC ATG CTG (SEQ IDNO:38), I99F: CCAGCGTATAATTCTAAGACA TTC TCC AAT GAC ATC ATG CTG (SEQ ID NO:39), I99R: CCAGCG TATAAT TCTAAG ACAAGA TCC AAT GAC ATC ATG CTG (SEQ IDNO:40), Yl 74A: GT GAG TCC TAC TTAAAAAAT GCTTTC GAC AAA GCC AAT GAGATA (SEQ ID NO:41).
  • the resulting plasmid was transformed into X33 cells and selected with Zeocin (Invitrogen, La Jolla CA). All granzyme B variants were expressed and purified to homogeneity by the wild-type protocol with yields between 0.5 and 3 mg/L. Activity was monitored in the supernatant using 1 ⁇ M Ac-AAD-pNA during expression.
  • the granzyme B variants were assayed for activity against Ac-IEPD-AMC (SEQ ID NO:32), and diluted in granzyme activity buffer (50 mM Na HEPES, pH 8.0, 100 mM NaCl, 0.01% Tween-20) to concentrations between 50 nM and 1 ⁇ M that yielded PSSCL activity levels approximately equal to 50 nM wild-type granzyme B.
  • granzyme activity buffer 50 mM Na HEPES, pH 8.0, 100 mM NaCl, 0.01% Tween-20
  • each variant was assayed in the Pl sub-library of a tetrapeptide completely diverse PSSCL (Youngchool Choe, in preparation).
  • Each sub-library consists of 19 pools containing 361 peptides, one each for the spatially addressed amino acids (cystine and methionine are deleted and D-alanine is included).
  • Enzymatic activity in the PSSCL was assayed for one hour at 30°C on a SpectraMAX Gemini fluorimetric plate reader (Molecular Devices Corporation, Sunnyvale, CA) and the rate of substrate hydrolysis analyzed with the
  • ACC or AMC leaving group using saturating amounts of wild-type granzyme B was exposed to saturating amounts of enzyme, and the total fluorescence measured over multiple days until all of the subsfrate was hydrolyzed. Individual kinetic measurements Individual kinetic measurements were performed using a SpectraMAX Gemini fluorometric plate reader. Each protease was diluted to between 50 nM and 1 ⁇ M in assay buffer. All ACC. substrates were diluted in DMSO to between 5 and 500 ⁇ M, while AMC subsfrates were diluted to between 20 and 2000 ⁇ M. Each assay contained less than 5% (v/v) DMSO.
  • Enzymatic activity was monitored every 15 seconds at excitation and emission wavelengths of 380 nm and 460 nm, respectively, for a total of 10 minutes. Rates of substrate hydrolysis were determined using the SOFTmax PRO data analysis software (version 3.1.1, Molecular Devices Corp.) and fit to the Michaelis-Menten equation using Kaleidagraph (Version 3.5, Synergy Software, Reading, PA). When the apparent K m values of for a substrate was greater than 2.2 ⁇ M, only k cat /K m was reported. The substrates were assayed for hydrolysis in duplicate at least twice by granzyme B and variants.
  • the variants were single mutants I99A, I99R, I99F, N218A, N218T and Y174A and double mutants N218A/I99A, N218A/R192A andN218A/R192E.
  • Initial activity in the complete diverse PSSCL indicated that all granzyme B variants specifically and uniquely hydrolyzed subsfrates containing Pl aspartic acid. This result is consistent with the strict specificity of granzyme B for aspartic acid.
  • PSSCL reveals extended specificity changes upon alanine mutagenesis Each alanine variant was profiled with the Pi-Asp AMC PSSCL.
  • Granzyme B I99A had a large effect on the PSSCL specificity profiles at the P2 position.
  • the mutation increased the hydrolysis of P2-Phe or Tyr substrates and reduced the hydrolysis of non-hydrophobic side chains without altering the P3 or P4 profiles compared to wild- type granzyme B (FIG. 1, Table 21).
  • Activity for the P2 amino acid Phe was nine times the activity for Pro compared to a 3.5 fold P2-Pro preference for wild-type granzyme B.
  • the novel hydrophobic preference also extends to tetrapeptide substrates.
  • the specificity constant, kc t /K ra for Ac-IEPD-AMC and Ac-IEFD-AMC were 57 M ' V 1 and 268 M ' V 1 , respectively, a five fold difference in activity.
  • N218A granzyme B did not change the P2 or P4 specificity profiles, but decreased the preference for PSSCL substrates with Glu at the P3 position, broadened the number of accepted P3 amino acids to include Ser and Ala, and increased the preference for Met and Gin (FIG. 1, Table 20). A similar result was observed for the R192K and R192A mutations to granzyme B .
  • Y174A granzyme B had activity approximately 10 fold less than the wild-type activity (470 M ' V 1 versus 3300 M ' V 1 ).
  • the decrease in activity was due to a decrease in the k cat value from 1.19 s "! to 0.87 s "1 and an increase in the K ⁇ from 370 ⁇ M to 1000 ⁇ M.
  • This reduction in activity with no change is specificity suggests that the phenyl side chain acts to stabilize the transition state and orient the scissile bond but has little effect on the identity of the P4 amino acid.
  • Table 20 shows the kinetic parameters for the hydrolysis of the individual tetrapeptide substrates Ac-IEPD-AMC (SEQ ID NO:32), Ac-IEFD-AMC (SEQ ID NO:35), Ac-IAPD-AMC (SEQ ID NO:42) and Ac-IKPD-AMC (SEQ ID NO:33), by the indicated Granzyme B protease.
  • the highlighted amino acids designate the target residues that deviate from the prefe ⁇ ed wild-type substrate.
  • Table 20 Granzyme B wild-type, I99A, Y174A and N218A mutein specificity.
  • the I99R, I99F, and N218T granzyme B variants were designed to test structural determinants found in the granzyme subfamily. Phe-99 occurs in the human homolog of rat granzyme B, and Arg-99 is found in granzymes A and C (FIG. 2, Table 21).
  • the I99F variant prefers the wild-type granzyme B substrate sequence Ile-Glu-Pro by the PSSCL, but the specificity for P4-Leu increases. The preference for P4-Ile versus P4-Leu is 2.6 fold as measured with tetrapeptide substrates for I99F granzyme B versus 370 fold for wild-type granzyme B (Table 21).
  • a decrease in the kc at , from 1.19 s "1 to 0.56 s "1 accounts for much of the reduction in activity.
  • a hydrophobic preference at P2 appears, but it is less apparent than the I99A variant.
  • Pro and Phe substrates are hydrolyzed at similar rates (kcat m is 330 M ' V 1 , 505 M ' V 1 , respectively).
  • the primary differentiation is in the K m values.
  • the K m of the P2-Phe substrate (980 ⁇ M) is less than the K m for the wild-type P2-Pro substrate (1800 ⁇ M), while the rates are equivalent (k ca t is 0.47 s "1 and 0.55 s "1 , respectively).
  • Asn218 faces the active site cleft and forms a hydrogen bond with the carboxylate group of Glu82 in ecotin. This central location arises from the truncation of the 220's loop compared to trypsin, and the kink induced by a cis-Pro at position 217. Asn218 was replaced with Thr, to probe the role of this hydrogen bond.
  • N218T decreased the preference for the PSSCL subsfrates with Glu at the P3 position, broadened the number of accepted P3 amino acids to include Ser and Ala, and increased the preference for Met and Gin (FIG. 2, Table 13).
  • the mutations did not change the P2 or P4 specificity.
  • Ala was the most prefe ⁇ ed P3 amino acid, and the preference for amino acids such as Lys remained low (k cat K m 600 M ' V 1 ) .
  • Kinetic rate constants for the individual tetrapeptide substrates Ac-IEPD-AMC (SEQ ID NO:32), Ac-IETD-AMC (SEQ ID NO:34), Ac-LEFD-AMC (SEQ ID NO:36) and Ac-LEPD-AMC (SEQ ID NO:37) illustrate the magnitudes of the altered specificity where the highlighted amino acids deviate from the prefe ⁇ ed wild-type substrate sequence. See Table 21. The presented ratios are of the wild-type preference (P) to the variant's novel preference (X). The novel preference (X) is the bold amino acid of the substrate. Ratios are shown for activity in the PSSCL libraries (PSSCL) and between individual tetrapeptide substrates (Activity). Table 21. Granzyme B wild-type, I99F, I99R and N218T mutein specificity.
  • N218 mutations in combination with 199 or R192 mutations dramatically alter specificity.
  • the N218A variant was shown to broaden the P3 specificity of granzyme B, so it was combined with two mutations at the structural determinant Argl 92.
  • the Rl 92A single mutation broadens the P3 specificity of rat granzyme B and results in a measured specificity constant of 55 M ' V ⁇ or Ac- IEPD-AMC, much lower than the nearly wild- type activity of the N218 variant (2200 M ' V 1 ).
  • the R192A N218A variant has no change at P2 and P4, an increased preference for Ala and Ser at P3 (FIG. 3, Table 22), and a slight preference for Glu over Lys.
  • the R192A variant prefers Glu to Lys by 7 fold whereas the R192A N218A variant is neutral within e ⁇ or.
  • the PSSCL results indicate a ⁇ 2 fold preference for Lys over Glu whereas the individual substrates indicate a 2 fold preference for Glu over Lys.
  • N218A/R192E completely reverses the P3 specificity from acidic to basic (FIG. 3, Table 22). From PSSCL results, the activity of P3-Glu versus Lys has been reversed to favor Lys by nine fold. The kinetic constants demonstrate a significant preference as well.
  • the specificity constants for N218A R192E for Ac-IEPD-AMC and Ac-IKPD-AMC are 10 M ' V 1 and 73 M ' V 1 , respectively (Table 22). This represents a seven fold preference for the basic amino acid, but a 330 fold reduction in activity compared to the wild-type preference for Ac-IEPD-AMC (3300 M ' V 1 versus 10 M ' V 1 ). The decrease in activity arises from an increase in the K m above the substrate concentration limit of 2 ⁇ M. The wild-type activity against the P3 -basic substrate Ac- IKPD-AMC is not improved with these mutations (220 M ' V 1 versus 73 M ' V 1 ).
  • the double mutation N218A R192E reverses the P3 specificity of rat granzyme B but the catalysis of P3-Lys substrates does not increase above 73 M ' V 1 .
  • Table 22 shows the kinetic constants for individual tetrapeptide substrates Ac-IEPD- AMC (SEQ ID NO:32), Ac-IKPD-AMC (SEQ ID NO:33) and Ac-IAPD-ACC (SEQ ID NO:42).
  • the preference for acidic amino acids at P3 is totally reversed with the R192E/N218A variant.
  • the highlighted amino acid in each substrates deviate from the prefe ⁇ ed wild-type substrate sequence.
  • the presented ratios are of the wild-type preference (P) to the variant's novel preference (X).
  • the novel preference (X) is the bold amino acid of the substrate. Ratios are shown for activity in the PSSCL libraries (PSSCL) and between individual tetrapeptide substrates (Activity). Table 22. Granzyme B WT, N218A/R192A and N218A/R192E mutein specificity.
  • the double mutation N218A/I99A had a dramatic effect on extended specificity altering the P2 through P4 specificity by PSSCL to the sequence Pro-Ala-Phe-Asp (FIG. 4, Table 23).
  • the P2 specificity is na ⁇ owed to prefer large hydrophobic amino acids, but in addition the P3 and P4 specificity have been broadened to include most aliphatic and ⁇ branched amino acids.
  • the activity of the N218 A/199 A variant is comparable to the activity of the 199 A variant against the Ac-IEFD-AMC substrate (270 M ' V 1 versus 290 M ' V 1 , respectively), though the tetrapeptide substrate with the highest specificity constant is Ac-IEFD-AMC (290 M ' V 1 ) rather than Ac-LEFD-AMC (37 M ' V 1 ).
  • This effect is due to an increase in the binding constant (890 ⁇ M versus >2000 ⁇ M), suggesting that some cooperativity in substrate preference is masked by the combinatorial nature of the libraries.
  • Table 23 shows the kinetic constants for the tefrapeptide substrates Ac-IEPD-AMC (SEQ ID NO:32), Ac-IEFD-AMC (SEQ ID NO:35), Ac-LEPD-AMC (SEQ ID NO:37), and Ac-LEFD-AMC (SEQ ID NO:36) and the calculated change in free energy compared to the wild-type sequence IEPD.
  • Table 23 Granzyme B wild-type and I99A/N218A mutein specificity
  • the difference in free energy will be positive if the wild-type substrate, IEPD, is a better substrate than the modified substrate.
  • IEPD wild-type substrate
  • the ⁇ G T * for P2-Pro versus P2-Phe is +0.71 and +1.1 kcal/mol when P4 is He or Leu, respectively (Table 24). The same result is observed for the P4 amino acid change for He to Leu ⁇ G T * +2.18 and 2.55 kcal/mol).
  • the N218 A 199 A variant is exhibiting a cooperative effect because the amount of binding energy contributed to the transition state stabilization from an ideal or non-ideal P2 amino acid is dependent on the identity of the P4 amino acid.
  • the granzyme B-like subfamily of serine proteases play an important role in granule-mediated immune responses. Clustered on chromosome 14 of human, rat and mouse species, they share active site architecture distinct from trypsin-like proteases.
  • Alanine is not found at amino acid 99 of any granzyme member or any trypsin fold serine protease, but other small amino acids such as Val and Gly are present. Arg is unique at position 99, found only in granzymes C and A. Both the human and mouse homologs of granzyme A have hydrophobic preferences at P2. Also of note is the increased P4-Leu preference in the I99F variant. This subtle difference between the rat (99-He), mouse (99-Phe), and human (99-Phe) homologues of granzyme B significantly alters small molecule inhibitor binding between species.
  • Argl92 is a determinant of extended specificity in granzyme B, activated protein C, thrombin, and Factor Xa. In granzyme B, P3 specificity is determined by both amino acids 218 and 192. Argl92 provides a strong electrostatic repulsion against Arg and Lys subsfrate side chains, while the polar Asn218 acts as a selector to exclude large hydrophobic and ⁇ branched amino acids. Asn218 serves as a steric determinant through the exclusion of large side chains from S3, and as a hydrogen bond donor for the wild-type Glu/Gln/Met preference.
  • the N218T variant was twice as efficient at cleaving the Ac-IEPD-AMC subsfrate as wild-type entirely due to an increase in the k cat of the protease.
  • the mutations at this position also decouple the interdependence between the P2 and P4 positions.
  • Cooperativity and interdependence between the extended substrate sites has been described in the serine protease subtilisin.
  • a favorable substrate amino acid at the Pl or P4 positions reduces the effect of a disfavored amino acid at another site.
  • An effective catalytic ceiling exists where the maximum amount of ground state substrate binding energy is converted to transition state stabilization and catalysis when P4 and Pl are ideal amino acids.
  • subtilisin hydrophobic, steric and structural effects all contribute to efficient catalysis of substrates by modulating the formation of the Michaelis complex. Mutations to subtilisin successfully alter the specificity and retain efficient catalytic rates. This is not the same for granzyme B, reflecting the fundamental difference between the their three-dimensional structures. The P4 site of WT granzyme B is energetically unlinked to the P2 site. The change in rate when the P2 substrate amino acid is varied is nearly independent of the identity of the P4 amino acid. Mutation of extended specificity determinants changed the relative importance and cooperative effects of extended specificity as observed for alanine substituted substrates against WT and N218A/I99A granzyme B.
  • subtilisin is a more flexible protease capable of adapting to mutagenesis that alters extended specificity without the equivalent reduction in activity, a situation where ground state binding is tightly coupled to access to the transition state.
  • the results of the systematic mutagenesis described here demonstrate that amino acids in direct contact with the extended subsfrate amino acids by side chain to side chain interactions contribute to the extended specificity of the enzyme. Mutation of the structural determinants changed the specificity and both the binding constant, K m , and the catalytic rate, k cat . Importantly, the hydrolysis by the variant proteases of their most preferred substrate was within 2 fold of the activity of the wild-type protease against the same subsfrate.
  • the variant proteases retain hydrolytic activity, binding constants and catalytic rates on the order of a nonideal wild-type substrate.
  • the alterations to specificity arise from the exclusion of non-ideal subsfrates rather than increased affinity of ideal substrates.
  • Position 99 has been well defined as a determinant of P2 specificity, and the combination of 192 and 218 defines P3.
  • Example 17 Muteins consisting of up to four mutations with increased selectivity towards VEGFR stalk region sequence, LVED. Multiple muteins were identified by PSSCL profiling with increased selectivity towards the LVED target cleavage sequence. They were grouped into sub-classes based on which subsite profile was most affected by the mutations: P4, P3 or P2. In addition to the muteins discussed in Example 16, mutations were found at four additional sites to have a role in the protease selectivity.
  • the serine protease MT-SPl has been chosen as scaffold protease for mutagenesis towards specific proteolysis of the VEGF and VEGFR in part because it has been well characterized with biochemical and stmctural techniques [Harris, Recent Results Cancer Res. 1998;152:341-52].
  • MT-SPl is a membrane bound serine protease with multiple extracellular protein- protein interaction domains.
  • the protease domain alone has been profiled using the totally diverse and Pl-Lys PSSCL (FIG. 19A-19C) revealing an extended specificity of (basic)-(non-basic)-Ser-Arg or (non-basic)-(basic)-Ser-Arg/Lys.
  • the X-ray crystallographic stmcture of MT-SPl reveals components proposed to regulate activity and a nine amino acid insertion in the 60's loop that may determine P2 specificity Variants of MT-SPl have been created and characterized.
  • Various protease muteins have been expressed and purified, as described below. Initial activity to verify activity and specificity have been performed, and sample results are provided in FIGS. 15-25.
  • a mutated MT-SPl polypeptide may contain a single mutation per polypeptide, or may contain two or more mutated residues in any combination provided as illustrated in Tables 17 and 18.
  • Wild-type and mutant MT-SPl are cloned into the pQE bacterial expression vector (Qiagen) containing an N-terminal 6 histidine tag, prodomain, and protease domain and the resulting constructs transformed into BL21 E. coli cells. Cells are grown in 100 mL cultures to an OD of 0.6 and expression of the protease in inclusion bodies is induced by adding IPTG to a final concentration of 1 mM.
  • the bacteria are pelleted by centrifugation and the pellet resuspended in 50 mM Tris pH 8, 500 mM KC1, and 10% glycerol (buffer A). Cells are lysed by sonication and pelleted by centrifugation at 6000x g. Pellets are resuspended in 50 mM Tris pH 8, 6 M urea, 100 mM NaCl and 1% 2-mercaptoethanol (buffer B). Membrane and organelles are pelleted by centrifugation at 10,000x g and the supernatant is passed over a nickel NTA column (Qiagen).
  • the column is washed with 50 mM Tris pH8, 6 M urea, 100 mM NaCl, 20 mM imidazole, 1% 2-mercaptoethanoland 0.01% Tween 20 (buffer D).
  • the column is washed again with buffer D without Tween 20.
  • the protease is then eluted from the column with 50 mM Tris pH 8, 6 M urea, 100 mM NaCl, 1 % 2-mercaptoethanol and 250 mM imidazole (buffer E).
  • the protease is then concentrated to a volume of ⁇ 1 mL and then dialyzed at 4°C overnight in 1 L of 50 mM Tris pH8, 3 M urea, 100 mM NaCl, 1% 2-mercaptoethanol, and 10% glycerol. Finally, the protease is dialyzed into 50 M Tris pH 8, 100 mM NaCl, and 10% glycerol at 4°C overnight. During the last dialysis step, the protease becomes autoactivated by self-cleavage resulting in the removal of the 6 histidine tag and prodomain. Result. Multimilligram quantities are obtained using bacterial expression system.
  • the protease is produced in inclusion bodies and is purified by a one-column purification procedure and then re-folded through successive dialysis steps (FIG. 15). Once refolded, the protease activates itself by cleavage at the juncture between the prodomain and the protease domain at the sequence RQAR/WGG.
  • Example 20 Determination of the Extended Specificity of MT-SPl Variants by PSSCL.
  • the Pl-Arg fixed PSSCL library is resuspended in DMSO and a ⁇ ayed in opaque black 96-well plates at a concentration of 5-10 nanomoles per well.
  • Variant proteases are diluted into 50 mM Tris pH 8, 50 mM NaCl, and 0.01% Tween 20 (MT-SPl activation buffer) at a concentration of 5 nM to 5 ⁇ M.
  • One hundred microliters of the protease solution is added to each well and fluorescence of the ACC leaving group is measured by excitation at 380 nm and emission at 460 nm using a Spectramax fluorescent plate reader (Molecular Devices).
  • the specificity of variant proteases at each of the P4-P2 extended subsites is determined by the fluorescence of each of the a ⁇ ayed amino acids in the P4-P2 PSSC libraries. Result. Screening by PSSCL confirms that wild-type MT-SPl has a preference for basic (Arg, Lys) at the P4 and P3 positions, in agreement with published data by Takeuchi et al, J. Biol. Chem., Vol. 275, Issue 34, 26333-26342, August 25, 2000. However, the PSSCL profile also reveals that its specificity is somewhat broad, such that a variety of amino acids will be accepted in the P4 and P3 positions in addition to Arg or Lys (FIG. 16 A).
  • the phagemid is constructed such that it (i) carries all the genes necessary for Ml 3 phage morphogenesis; (ii) it carries a packaging signal which interacts with the phage origin of replication to initiate production of single-stranded DNA; (iii) it carries a dismpted phage origin of replication; and (iv) it carries an ampicillin resistance gene.
  • the combination of an inefficient phage origin of replication and an intact plasmid origin of replication favors propagation of the vector in the host bacterium as a plasmid (as RF, replicating form, DNA) rather than as a phage. It can therefore be maintained without killing the host.
  • a plasmid origin means that it can replicate independent of the efficient phage-like propagation of the phagemid.
  • the vector can be amplified, which in turn increases packaging of phagemid DNA into phage particles.
  • Fusion of the MT-SPl variant gene to either the gene 3 or gene 8 Ml 3 coat proteins can be constructed using standard cloning methods. (Sidhu, Methods in
  • a combinatorial library of variants within the gene encoding MT-SPl is then displayed on the surface of Ml 3 as a fusion to the p3 or ⁇ 8 Ml 3 coat proteins and panned against an immobilized, aldehyde-containing peptide co ⁇ esponding to the target cleavage of interest.
  • the aldehyde moiety will inhibit the ability of the protease to cleave the scissile bond of the protease, however, this moiety does not interfere with protease recognition of the peptide.
  • Variant protease-displayed phage with specificity for the immobilized target peptide will bind to target peptide coated plates, whereas non-specific phage will be washed away.
  • proteases with enhanced specificity towards the target sequence can be isolated.
  • the target sequence can then be synthesized without the aldehyde and isolated phage can be tested for specific hydrolysis of the peptide.
  • Example 22 Identification of MT-SPl mutein cleavage in the stalk region of VEGFR2
  • the polypeptide sequence of VEGF receptor 2 (VEGF-R2/KDR), showing the respective sequences of the extracellular (SEQ ID NO:38) and intracellular (SEQ ID NO: 39) domains, is provided in Table 12. Sequences that closely match the P4-P1 native subsfrate specificity of MT-SPl are shown in bold. Two sequences match the recognition profile of both L172D and wild-type MT-SPl: the boxed sequence RVRK and the double underlined sequence RRVR. Table 24. VEGFR2 KDR Substrate Specificity of Targeted MT-SPl
  • VEGF-R2 VEGF-R2 fused to the Fc domain of mouse IgG (2.5 ⁇ g) was resuspended with 1 ⁇ M MT-SPl and variant proteases in 17.1 uL of MT-SPl activation buffer. The reaction was incubated at 37°C for 2 hours, deglycosylated with PNGaseF, and separated by SDS-PAGE elecfrophoresis. Full length Flkl-Fc and cleavage products were identified by staining with Coomassie brilliant blue and the N-termini sequenced by the Edman protocol.
  • Purified VEGFR2-Fc is cleaved by wild-type and mutant MT-SPl at the sequence RRVR KEDE in the extracellular stalk region of the receptor.
  • the present invention provides proteases that can cleave the VEGFR in the stalk region, and in one embodiment of the invention, such proteases are administered to a patient in need of treatment for cancer, macular degeneration, or another disease in which angiogenesis plays a causative or contributive role.
  • VEGFR2-Fc is efficiently cleaved by wild-type and mutant MT-SPl (FIG. 18).
  • Cleavage by variant proteases yields cleavage products with apparent molecular weights of ⁇ 80 kDa and 30 kDa; analysis of potential cleavage sites in VEGFR2 suggests that MT-SPl variants target the stalk (membrane proximal) region of VEGFR2.
  • the mutant L172D cleaves full-length VEGFR2 but at a reduced rate compared to the wild-type.
  • Several mutants ( ⁇ 175D and D217F) cleave the receptor with higher efficiency than wild-type. None of the protease variants or wild-type cleave the Fc domain.
  • Example 24 Assaying for cleavage of VEGF receptor from endothelial cells.
  • Human umbilical vein endothelial cells were purchased from Cambrex and cultured in EBM-2 (endothelial cell basal medium, Cambrex) with full supplements including 2% fetal calf semm (FCS) and antimycotics-antibiotics.
  • EBM-2 endothelial cell basal medium, Cambrex
  • FCS fetal calf semm
  • VEGF was added at a final concentration of 20 ng/mL and the cells were incubated for 72 hours. At the end of the 72 hours, cell count was determined by MTT assay (Sigma) according to the manufacturer's protocol.
  • MTT assay Sigma
  • HUVECs were grown in 24-well plates and treated with proteases as described above. After 3 hours incubation, 100 uL of media was removed and the protease inhibitor Pefabloc (Roche) was added to a final concenfration of 1 mg mL. The media was then added to Maxisorp plates (Nunc) that had been treated with a monoclonal antibody recognizing the extracellular domain of VEGFR2 (MAB3573, R & D Systems, 1:125 dilution in PBS).
  • FIG 21A Wild-type MT-SPl and the more specific mutants, including CBl 8, CB83 and CB 152, efficiently inhibited VEGF-dependent proliferation of endothelial cells in a dose-dependent manner.
  • Figure 2 IB shows that the MT-SPl variants cleave the VEGF receptor on the surface of endothelial cells. Shown is a Western blot in which HUVECs are incubated with the buffer control or MT-SPl variants, and then cell extract are probed with an antibody recognizing the intracellular domain of VEGFR2.
  • Wild-type MT-SPl and variants cleave the full-length receptor (upper band) to generate a truncated form (lower band).
  • the extracellular domain (ectodomain) of the cleaved receptor can be detected in the media, as shown by the ELISA in Figure 21C; the released ectodomain is detectable in samples treated with MT-SPl and variants, but not in the control.
  • Example 25 Cornea Micropocket Model. To determine the acute maximum tolerated dose, escalating doses of purified wild-type and variant MT-SPls were injected i.v into C57BL/6 mice. The mice were observed for outward signs of toxicity, and death.
  • a comeal micropocket was dissected toward the limbus with a von Graefe knife #3 (2 x 30 mm), followed by pellet implantation and application of topical erythromycin. After 8 days, neovascularization is quantitated by using a slit lamp biomicroscope and the formula 2 ⁇ x (vessel length/10) (clock hours). P values were determined by using a two-tailed t test assuming unequal variances (Microsoft EXCEL). Varying doses of proteases were injected by i.p. twice a day at 12 hour intervals starting at day 0 until day 7. Results.
  • Wild-type MT-SPl was well tolerated by mice, with an acute maximum tolerated dose (MTD) determined to be 50 mg/kg (FIG 22).
  • MTD acute maximum tolerated dose
  • some of the MT-SPl variants that were shown to have nanower selectivity in the profiling libraries (see FIG 16) were better tolerated (i.e. had lower toxicities), resulting in higher maximum tolerated doses.
  • CB 18 and CB 152, for instance, were tolerated at doses that resulted in death for wild-type MT-SPl . This demonstrates that nanowing the selectivity can be a mechanism for reducing the toxicity of protease dmgs.
  • Wild-type MT-SPl and variants were tested for their ability to inhibit VEGF- induced angiogenesis in the mouse cornea micropocket model. As outlined above, a pellet of VEGF was implanted into the cornea of mice, which is normally avascular, and the amount of neovascularization was quantitated after 8 days. When mice were treated with either wild-type or variant MT-SPl, neovascularization was inhibited in a dose dependent manner (FIG. 24). Treatment of mice with wild-type MT-SPl at the MTD (50 mg/kg) resulted in 42% inhibition of neovascularization.
  • Example 26 Miles Assay for Vascular Permeability.
  • VEGF In addition to angiogenesis, VEGF also induces the permeability of blood vessels, resulting in the leakage of fluids into the su ⁇ ounding tissue. NEGF-induced vascular permeability was measured using the Miles assay.
  • nude (athymic) mice were injected with 0.5% Evan's blue dye (100 uL in PBS, Sigma) by tail vein injection.
  • 100 ng of NEGF in 20 uL PBS was injected infradermally into the back of the mice in duplicate spots.
  • Vascular permeability is visualized by the appearance of blue spots at the site of VEGF injection due to the leakage of the dye. The extent of vascular permeability can be measured semi-quantitatively by measuring the area of the blue spots. To determine if they inhibited vascular permeability, wild-type MT-SPl and variants were injected i.p.
  • Murine Lewis lung carcinoma (LLC) cells are passaged on the dorsal midline of C57BL/6 mice or in DMEM/10% FCS/ ⁇ enicillin/sfreptomycin(PNS)/L-glutamine.
  • T241 murine fibrosarcoma is grown in DMEM/10% FCS PNS/L-glutamine and human pancreatic BxPc3 adenocarcinoma in RPMI medium 1640/10% FCS/PNS.
  • Tumor cells (10 6 ) are injected s.c.
  • Tumor size in mm 3 is calculated by caliper measurements over a 10- to 14-day period by using the formula 0.52 x length (mm) x width (mm), using width as the smaller dimension. See, e.g., Kuo et al., PNAS, 2001, 98:4605-4610.
  • scaffold proteases and variants have been successfully expressed as active proteases in yeast or bacterial expression systems at multi-milligram quantities. See, e.g., protocols described in Harris 1998 and Takeuchi, 2000.
  • MT-SPl was engineered to obtain muteins that selectively cleave Flk-l/KDR. Additional MT-SPl muteins, shown in Table 17, where cloned and expressed as described above.
  • MT-SPl variants were expressed in bacteria and purified from inclusion bodies. Each protease retains high catalytic activity and is >99% pure making them appropriate for crystallographic studies.
  • Table 25 depicts the potential target cleavage sequences for wild-type and mutein
  • MT-SPl represents any hydrophobic amino acid (i.e. glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, or tryptophan) and "Xxx” represents any amino acid. "Xxx” represents any amino acid.
  • Example 29 Muteins consisting of one, two and three mutations with increased selectivity towards VEGFR stalk region sequence, RRVR
  • Mutations such as Phe99 to Trp, Asn, Asp, Ala, or Arg increased the P2 selectivity for Ala, Ser, Trp, Lys and He containing subsfrates. Additional mutations that affected the P2 selectivity were Metl 80 to Glu and Ala and Trp215 to Tyr and Phe. Mutation of Glnl92 to Arg and Glu altered the P3 selectivity alone. Mutations at Tyr 146 (Asp), Leul72 (Asp), Glnl75 (Asp), Lys224 (Phe), and Metl80 (Glu) increased the selectivity of the variants, towards both P3 and P4 Arg and Lys containing subsfrates as in variant L172D (CBl 8) (FIG 16B).
  • variant proteases with highly selective P3 and P4 profiles such as the variants L172D/Q175D (CB83) and Y146D/K224F (CB155) (FIG 162E&F). Results.
  • mutations identified individually to na ⁇ ow the protease selectivity at P2 and at P3/P4 multiple variants were made that had greater than four fold selectivity towards Arg and Lys residues at the P3 and P4 positions, and altered P2 specificity.
  • the second sequence, RQAR is a prefe ⁇ ed sequence of MT-SPl as determined by subsfrate profiling. It also matches the sequence in the full length protease that must be cleaved for protease activation. Michealis-Menton kinetic constants were determined by the standard kinetic methods.
  • the subsfrate is diluted in a series of 12 concenfrations between 1 mM and 2 ⁇ M in 50 ⁇ L total volume of MT-SPl activity buffer in the wells of a Costar 96 well black half-area assay plate.
  • the solution is warmed to 30°C for five minutes, and 50 ⁇ L of a protease solution between 0.1 and 20 nM was added to the wells of the assay.
  • the fluorescence was measured in a fluorescence spectrophotometer (Molecular Devices Gemini XPS) at an excitation wavelength of 380 nm, an emission wavelength of 450 nm and using a cut-off filter ser at 435 nm.
  • the rate of increase in fluorescence was measured over 30 minutes with readings taken at 30 second intervals.
  • the specificity constant (l Km) is a measure of how well a substrate is cut by a particular protease.
  • Mutant proteases that match the desired specificity profiles, as determined for example, by subsfrate libraries, are assayed using individual peptide subsfrates co ⁇ esponding to the desired cleavage sequence. Individual kinetic measurements are performed using a Spectra-Max Delta fluorimeter (Molecular Devices). Each protease is diluted to between 50 nM and 1 ⁇ M in assay buffer. All ACC substrates are diluted with MeSO to between 5 and 500 ⁇ M, while AMC subsfrates are diluted to between 20 and 2000 ⁇ M. Each assay contain less than 5% MeSO. Enzymatic activity is monitored every 15 seconds at excitation and emission wavelengths of 380 nm and 460 nm, respectively, for a total of 10 minutes. All assays are performed in 1% DMSO.
  • Example 32 Screening for cleavage of full-length proteins
  • Variant proteases are assayed to ascertain that they will cleave the desired sequence when presented in the context of the full-length protein, and the activity of the target protein is assayed to verify that its function has been destroyed by the cleavage event.
  • the cleavage event is monitored by SDS-PAGE after incubating the purified full- length protein with the variant protease.
  • the protein is visualized using standard Coomasie blue staining, by autoradiography using radio labeled protein, or by Western blot using the appropriate antibody.
  • the target protein is a cell surface receptor, cells expressing the target protein are exposed to the variant protease.
  • VEGF Vascular endothelial growth factor
  • VEGFR- 1 /Fit- 1 VEGF-2/KDR
  • VEGFR-3/Flt-4 Three high affinity cognate receptors to VEGF have been identified: VEGFR- 1 /Fit- 1 , VEGFR-2/KDR, and VEGFR-3/Flt-4.
  • VEGF 165 a 165 amino acid recombinant version of VEGF, VEGF 165 , was assayed by SDS-PAGE.
  • VEGF 165 was reconstituted in PBS to a concenfration of 0.2 ⁇ g/ ⁇ L and diluted to a final concentration of 5 ⁇ M. Solutions with no protease and lOOnM MT-SPl or CB 152 were incubated with the VEGF at 37°C for five hours. The resulting protein cleavage products were deglycosylated, separated by SDS-PAGE, and silver stained (FIG. 26).
  • MT-SPl efficiently cleaves VEGF 165 under the assay conditions while the more selective variant CBl 52 does not.
  • CBl 52 a variant with na ⁇ ow selectivity to the RRVR sequence in the stalk region of VEGFR2
  • CBl 52 does not cleave VEGF, but does cleave VEGFR and can be dosed at higher concenfrations due to reduced toxicity. Cleavage Of VEGFR.
  • 125 I-VEGFR (40,000 cpm) is incubated with varying concenfrations of protease, samples are boiled in SDS-PAGE sample buffer and examined on a 12% polyacrylamide gel. The gels are dried and exposed to x-ray film(Kodak) at -70 °C.
  • VEGFR Binding Assay 125 I- VEGFR or PMN are incubated with varying concentrations of proteases as above. The binding of 125 I- VEGFR exposed to proteases to normal PMN, or the binding of normal 125 I- VEGFR to PMN exposed to proteases, are quantified using scintillation.
  • MT-SPl and trypsin were assayed in the presence of increasing concentrations of fetal calf semm.
  • the high concentrations of macromolecular protease inhibitors present in semm makes it a good in vitro system to test whether a protease would be active in vivo.
  • MT-SPl and trypsin were resuspended in Dulbecco's Modified Eagle's Medium (DMEM) at 100 nM and 80 nM, respectively, with increasing semm concenfrations (0-10%) in a final volume of 100 ⁇ L.
  • DMEM Dulbecco's Modified Eagle's Medium
  • a fluorogenic peptide substrate (Leu-Val-Arg-aminomethylcoumarin) was added to a final concenfration of 15 ⁇ M and fluorescence was detected in a fluorescence plate reader (Molecular Devices) with an excitation wavelength of 380 nm and an emission wavelength of 460 nm.
  • trypsin shows very strong activity in 0% semm, with the enzyme using up all the substrate after -400 seconds.
  • trypsin activity is drastically reduced, presumably due to the binding of macromolecular protease inhibitors.
  • MT-SPl shows virtually the same activity in all concenfrations of semm, suggestive that there are no endogenous protease inhibitors in semm that inactivate MT-SPl .

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Abstract

La présente invention a trait à des procédés pour la création et l'utilisation de protéases de mutéine à spécificité modifiée pour des molécules cibles dont elles assurent le clivage. L'invention a également trait à des procédés d'utilisation de granzyme B de type sauvage et de mutéine pour le ciblage de protéines de récepteur VEGF pour le traitement de maladies, tells que le cancer. Le clivage de récepteur VEGF au niveau de certaines séquences substrat avec des protéases de granzyme B de type sauvage et de mutéine constitue un procédé pour le traitement de pathologies associées à l'angiogenèse.
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WO2021030787A1 (fr) 2019-08-15 2021-02-18 Catalyst Biosciences, Inc. Polypeptides de facteur vii modifiés pour une administration sous-cutanée et un traitement à la demande
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