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WO2019032472A1 - Inhibiteur de matriptase modifié par haute affinité - Google Patents

Inhibiteur de matriptase modifié par haute affinité Download PDF

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Publication number
WO2019032472A1
WO2019032472A1 PCT/US2018/045431 US2018045431W WO2019032472A1 WO 2019032472 A1 WO2019032472 A1 WO 2019032472A1 US 2018045431 W US2018045431 W US 2018045431W WO 2019032472 A1 WO2019032472 A1 WO 2019032472A1
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Prior art keywords
matriptase
polypeptide
cancer
protein
domain
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Aaron MITCHELL
Jennifer R. Cochran
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Leland Stanford Junior University
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Leland Stanford Junior University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1833—Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8114—Kunitz type inhibitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/81—Protease inhibitors
    • G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • G01N2333/811—Serine protease (E.C. 3.4.21) inhibitors

Definitions

  • Matriptase is also known to activate other proteases and growth factors including urokinase plasminogen activator (uPA) (11), pro-macrophage stimulating protein (Pro-MSP) (13), and platelet derived growth factor-D (PDGF-D) (14), all of which play key roles in cancer growth and metastasis.
  • uPA urokinase plasminogen activator
  • Pro-MSP pro-macrophage stimulating protein
  • PDGF-D platelet derived growth factor-D
  • matriptase has been identified as a critical driver of other diseases, including iron overload disease (15) and osteoarthritis (16), and has been shown to activate the human airway influenza virus (H1N1) (17) and human immunodeficiency virus (HIV) (18).
  • matriptase overexpression Although the correlation of matriptase overexpression, dysregulation, and disease progression is well-established, effective matriptase inhibitors are lacking, highlighting an important clinical need.
  • HAI-1 hepatocyte growth factor activator inhibitor type-1
  • HGFA hepatocyte growth factor activator
  • HGFA hepatocyte growth factor activator
  • hepsin 28, 29
  • kallakrein-4/5 9, 30
  • the balance between substrate activation and protease inhibition is thought to be critical to the metastatic potential of tumor cells (Fig. 1 B).
  • the ratio of HAI-1 expression to matriptase expression correlates with cancer aggression and patient prognosis, and has been established as a key biomarker (2, 5, 7, 10, 31, 32).
  • KD1 domain (approximately 6 kDa vs 58 kDa for HAI-1) confers a short circulating half-life of 20 minutes, which greatly limits its therapeutic efficacy. While chemical conjugation of KD1 to polyethylene glycol (PEG) showed significant extension in serum half-life (35), this approach does not further improve the inhibition constant beyond wild-type KD1.
  • PEG polyethylene glycol
  • the polypeptide may comprise a first Kunitz domain that is at least 90% identical to the entire contiguous length of the KD2/1 fusion of SEQ ID NO: 1.
  • the polypeptide is believed to bind to matriptase with an affinity that is sufficient to effectively outcompete pro-HGF substrate activation and has a half-life in serum that is sufficient to mitigate the need for frequent dosing. Further, because some embodiments of the polypeptide comprise a chimeric domain made from only human sequences, it is expected that the polypeptide may be well tolerated immunologically. In some embodiments, the polypeptide may be comprised within a larger protein which, in some embodiments, may be Hepatocyte growth factor activator inhibitor- 1 (HAI- 1) or a variant thereof.
  • HAI- 1 Hepatocyte growth factor activator inhibitor- 1
  • This polypeptide further comprises multimerization domain, e.g., an Fc domain to increase valency of the Kunitz domains in the protein and increases the half- life of the protein in the blood.
  • multimerization domain e.g., an Fc domain to increase valency of the Kunitz domains in the protein and increases the half- life of the protein in the blood.
  • such a polypeptide should contain 4 matriptase binding sites, which increases matriptase binding compared to KD1 and KD2/1 alone.
  • a method for inhibiting a matriptase protease is also provided. This method may comprise contacting the matriptase protease with the present polypeptide, thereby inhibiting the matriptase protease.
  • a method of treating a matriptase-related disease or condition is also provided.
  • this method may comprise administering a therapeutically effective amount of the present polypeptide the subject.
  • the polypeptide finds particular use in treating cancer, iron overload disease, osteoarthritis, influenza or human immunodeficiency virus. In some embodiments, the polypeptide inhibits cancer progression in the subject.
  • the biosensor is a fusion protein and comprises, in order: an N-terminal domain of a reporter protein; a cleavage site for a protease; and a C-terminal domain of the reporter protein.
  • the fusion protein emits an optically-detectable signal and cleavage of the fusion protein by the protease abolishes the optically-detectable signal. Screening methods that employ the biosensor are also provided.
  • Fig. 1 Schematic of the HAI-1 inhibitor, which naturally regulates matriptase activity and levels of Pro-HGF activation, thus preventing cancer progression in healthy tissue.
  • B Biological representation of the tumor environment. Dysregulated matriptase cleaves Pro-HGF into activated HGF, which is competent to bind to and stimulate its cognate receptor, c-Met. Ligand-receptor dimerization then triggers intracellular signaling pathways that in turn stimulate cellular phenotypic responses, including cell growth, proliferation, and migration.
  • FIG. 2 (A) Library screening identifies a chimera of KDl and KD2 that binds matriptase. Representative FACS plots are shown from separate yeast library sorting rounds, including sorting gates used to isolate phenotypically improved KD2 variants. (B) Sequence alignment of clone 33, which is a chimera of KD2/KD1, (termed KD2/1) with KD2 graft 2, wild-type KDl, and shared sequence space of KDl and KD2.
  • Fig. 3 Schematic depiction of the HAI- 1 based protease inhibitor panel engineered to contain varying functional domains.
  • Engineered domains include KD1-R260A, which contains a point mutation that disrupts matriptase binding, and the chimeric domain KD2/1.
  • Several constructs are genetically fused to the Fc region of an antibody domain to increase valency and molecular size.
  • Fig. 4. Ability of soluble inhibitors to inhibit matriptase activity on PC3 (prostate),
  • MDA-MB-231 (breast), and A549 (lung) cancer cells.
  • A Dose response of normalized matriptase inhibition. Mean IC50 values reported with log standard deviation. Wild-type HAI-1 monomer; wild-type KDl monomer; KD1-KD2/1-Fc.
  • B Effects of KD1-KD2/1-Fc on cancer cell invasion in the presence of human embryonic kidney (HEK) cells or HEK cells transfected to overexpress pro-hepatocyte growth factor (HEK(-i-ProHGF)). Cancer cells alone represent negative controls. Significance quantified with pair wise ⁇ -test;
  • Fig. 5 The full length HAI-1 protein is comprised of the N-terminal domain, the internal domain, the first kunitz domain, the LDL domain, the second kunitz domain, the transmembrane domain, and the intracellular domains. Sequences of KDl -wild- type and KD2-wild-type; the boxed region is the matriptase binding interface of KDl (Arg-Cys-Arg- Gly) (SEQ ID NO: 16) which is absent in KD2.
  • HAI-1 Hemagglutinin- HA or cmyc
  • Binding profiles may vary due to the polyclonal nature of the HAI- 1 primary antibody.
  • D The matriptase binding profile of each displayed domain from at least 10,000 yeast cells (in duplicate) was quantified and presented relative to KDl wild-type binding. From top to bottom, SEQ ID NOs.: 4-7.
  • Fig. 6 Individual clones from the sequence summary table were transformed into yeast and tested for expression and binding to 10 nM matriptase. Binding was quantified and compared to KDl wild-type (positive binding control) and KD2 wild-type (negative binding control). Only clone 33 (KD2/KD1) bound to matriptase at levels significantly greater than KD2 wild-type. Data represents average binding fluorescence quantified from at least 10,000 yeast cells.
  • Fig. 7 Fast protein liquid chromatography (FPLC) chromatograms (A-C) of purified proteins. All proteins were able to be recombinantly expressed except KDlx2-Fc which revealed no chromatogram absorbance peak (C) and trace protein bands on SDS-PAGE (D) and quickly degraded over time preventing functional assessment. Size exclusion chromatograms represent elution time (min) versus protein absorbance at 280nm.
  • FPLC Fast protein liquid chromatography
  • Chromatograms are representative of at least three separate protein productions and purification procedures.
  • Fig. 8 Dose response plots measuring the effects of (A) each inhibitor on matriptase activity with full panel of inhibitor concentrations (left) or initial inhibitor concentrations (right), and protease activity of (B) KD1-KD2/1-Fc (C) KD1 wild-type monomer (D) HAI-1 wild-type monomer.
  • Fig. 9 Inhibition modality of KD1-KD2/1-Fc was tested and compared with KD1 wild-type monomer. Initial velocity of matriptase activity was plotted against substrate concentration for each inhibitor concentration tested. Plots were then fit to a Michealis- Menten curve to derive Km and Vmax values. Competitive inhibition modality is characterized by an increasing Km value and constant Vmax value, with increasing inhibitor concentration. Mean and standard deviation values reported.
  • Fig. 10 Schematic of huPro-HGF which includes a five domain a-chain (N- terminal domain and four Kringle (K) domains), the ⁇ -chain containing the serine protease homolog (SPH) domain, and a C-terminal histidine (His) tag.
  • the matriptase recognition and cleavage site (red arrow) is located between the huPro-HGF a and ⁇ chains.
  • the histidine tag enables detection in a western blot to confirm molecular weight of approximately 80 kDa (non-reduced), also verified by FPLC.
  • Reaction products from (C) were then added to (D) cultured Madin-Darby Canine Kidney (MDCK) cells to qualitatively measure the activity of pro-HGF in the presence of increasing inhibitor concentration.
  • Activated pro-HGF is indicated by increased cell scattering, while greater matriptase inhibition and less active pro- HGF is indicated by reduced cell migration.
  • Fig. 11 (A) KD1-KD2/1-FC (250nM) binding to matriptase expressed on cancer cells was quantified by flow cytometry. In parallel, a human matriptase specific antibody was used to measure matriptase expression on each cancer cell line: A549, PC3, MDA-MB-231. Secondary antibody only served as a negative control. (B) Inhibition by KD1-KD2/1-Fc on cancer cell expressed matriptase activity was quantified by incubating inhibitor with each cancer cell line and measuring product emission over time on a kinetic plate reader.
  • Fig. 12 is a table providing a summary of representative mutants isolated from the seventh round of sorting.
  • KD2 variant amino acid sequences were analyzed and compared with the library starting sequence KD2-graft 2 and KDl wild- type.
  • Clone 33 is a chimera of KD2 and KDl (termed KD2/1) that contains 6 amino acids exclusive to KD2 at the N- terminus, followed by the KDl exclusive sequence regions. The original sequence shared between KDl and KD2 is highlighted. Underlined residues in KD2/1 indicate positions where a nucleotide change was found that did not result in an amino acid mutation.
  • KD2- graft 2 SEQ ID NO: 1
  • KD1-WT SEQ ID NO: 3
  • Fig. 13 is a table showing the sequences of KD2/1 (top row-chimera), annotated KD2/1 sequence (middle row), and wild-type KDl (bottom row); along with residue position numbers. Highlighted regions are as follows: KD2 domain exclusive amino acids (light green), KDl domain exclusive amino acids, cysteine residues, flexible glycine/serine/valine residues, primary matriptase binding residues, secondary matriptase binding residues, secondary protein structures (beta sheet-light; alpha helix), and loop regions.
  • KD2/1 SEQ ID NO: 2;
  • Fig. 14 is a table showing the summary of the sequences of variants isolated from
  • the table is arranged with the KDl WT reference sequence as the first row, followed by variants from each sort specified. Consensus mutations are highlighted, with similar positional residue changes highlighted in the same color. KDl WT sequences are highlighted. Sequenced variant: SEQ ID NO: 3.
  • Fig. 15 is a table showing the summary of the sequences of variants isolated from KDl Library; Round 2; Sort 4.
  • the table is arranged with the KDl WT reference sequence as the first row, followed by variants from each sort specified. Consensus mutations are highlighted, with similar positional residue changes highlighted.
  • Sequenced variant SEQ ID NO: 3.
  • Fig. 16 shows a matriptase biosensor design.
  • A ddRFP-based biosensor schematic. Following proteolytic linker cleavage, the fluorescent "A" copy separates from the stabilizing "B” copy, resulting in loss of "A” copy fluorescence.
  • B Example time course plot of biosensor mechanism; addition of matriptase cleaves the biosensor and reduces fluorescence over time, while absence of matriptase retains fluorescence over time.
  • C Matriptase cleavable linker designs and sequence information. Design name is derived from number of amino acids flanking the scissile bond.
  • Scissile bond is highlighted (Arg) and (Val) and is derived from the natural pro-macrophage stimulating protein (Pro-MSP) sequence.
  • Fig. 17 shows biosensor kinetic measurements and characterization.
  • A Time course trajectories of biosensors B3 to B IO with matriptase and
  • B velocity graphs in the presence of varying concentrations of matriptase (Matr.).
  • Fig. 18 shows the application of B4 for measuring protease activity and inhibition.
  • A B4 measurement of matriptase activity expressed on human A549 lung, PC3 prostate, and MDA-MB-231 breast cancer cell lines, compared with media alone; *p ⁇ 0.01 vs cell condition.
  • B B4 measurement of matriptase inhibition by soluble KD1 inhibitor.
  • C B4 measurement of matriptase inhibition by yeast-displayed wild-type KD1, KD1-R260A, or non-induced yeast controls with or without matriptase.
  • Fig. 19 provides schematics of matriptase and HAI-1 inhibitor protein.
  • FIG. 1 Illustrated schematic of the matriptase mediated activation of Pro-HGF and inhibition by HAI-1.
  • FIG. 20 Illustrated schematic of the HAI-1 inhibitor protein; including extracellular and intracellular domain regions.
  • the full length HAI-1 protein is comprised of the N-terminal domain, the internal domain, the first kunitz domain, the LDL domain, the second kunitz domain, the transmembrane domain and the intracellular domains.
  • KDl and KD2 are sub domains of the full length HAI-1 protein. KDl has been demonstrated to be the only binding domain of HAI-1, while KD2 has been shown to not have any binding capacity for Matriptase.
  • Fig. 20 shows directed evolution by yeast surface display, KDl Round 1 and 2 Sort
  • Fig. 21 shows a sequence summary from KDl library, Round 1. Sequence summary of variants isolated from KDl Library Round 1, sort 5 and sort 6. The table is arranged with the KDl WT reference sequence as the first row, followed by variants from each sort specified. Consensus mutations are highlighted, with similar positional residue changes highlighted. KDl WT sequences are highlighted. Sequenced variant: SEQ ID NO: 3.
  • Fig. 22 shows a sequence summary from KDl library, round 2. Sequence summary of variants isolated from KDl Library; Round 2; Sort 4. The table is arranged with the KDl WT reference sequence as the first row, followed by variants from each sort specified. Consensus mutations are highlighted, with similar positional residue changes highlighted in. Sequenced variant: SEQ ID NO: 3.
  • Fig. 23 shows the KDl Structure In Complex With Matriptase With KDl Mutation Summary from Sort Round 1 and 2.
  • Crystal structure Pymol (PDB_4ISO) images of KDl (red) in complex with matriptase (green) reveal the (A) mutation locations of consensus mutations recovered from the KDl Library Sort Round 1 and KDl Library Sort Round 2: S253; N254; S277; L284; 1297. Closer zoom reveals the location of the most prevalent mutation from KDl Library Sort Round 2, S277R, combined with either (B) N254Y or (C) N254K. Mutation side chains in (B) and (C) are highlighted as described above. Dotted line indicates proximity for hydrogen bond formation by S277R.
  • Fig. 24 shows yeast surface display characterization of KDl variants.
  • Wild- type (WT) KDl and KDl variant domains were characterized as yeast displayed domains for their expression levels and matriptase binding compared with WT KDl.
  • A Matriptase binding affinity (Kd) was measured for each yeast displayed variant using a direct, equilibrium binding assay with increasing concentration of soluble matriptase.
  • B Yeast display expression levels were measured for select KDl variants and compared with WT KDl.
  • C Competitive binding results of matriptase binding in the presence of increasing substrate, values are the quantified concentration of substrate required to reduce matriptase binding 50% (IC50).
  • Fig. 25 shows soluble wild- type KDl Fc fusion inhibitor construct designs and production.
  • Soluble KDl based matriptase inhibitor Fc fusion construct (A) illustrations and (B) size exclusion chromatograms.
  • Fig. 26 shows soluble KDl variant inhibitor construct designs and production.
  • Soluble KDl based matriptase inhibitor construct (A) illustrations and (B) size exclusion chromatograms, including the KDl engineered N254Y and S277R variants in KDl domain (i), full length (ii), and Fc formats (iii).
  • Fig. 27 shows matriptase inhibition characterization of soluble KDl variants.
  • Fig. 28 shows yeast surface display and matriptase binding characterization of HAI- 1 domains.
  • Yeast surface display of HAI- 1 proteins and characterization (A) Schematic depiction of yeast surface display, a powerful protein engineering tool used to measure protease-inhibitor interactions.
  • B The matriptase binding profile comparing the matriptase binding potential of WT KD2 (non-binding control) to the WT KD1 domain (positive binding); FACS plots represent 10,000 yeast cells.
  • C Yeast displayed HAI-1 based inhibitors were also characterized for their ability to be expressed and folded on the yeast surface.
  • FIG. 29 shows a schematic overview of directed evolution by yeast surface display and affinity maturation of the KD1 library. Process overview of library mutagenesis and yeast surface display sorting procedure.
  • nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.
  • malignant is meant to refer to a tissue, cell or organ which contains a neoplasm or tumor that is cancerous as opposed to benign.
  • Malignant cells typically involve growth that infiltrates tissue (e.g., metastases).
  • benign is meant an abnormal growth which does not spread by metastasis or infiltration of the tissue.
  • a malignant cell can be of any tissue, e.g., epithelial.
  • tumor progression or “tumor metastasis” is meant the ability of a tumor to develop secondary tumors at a site remote from the primary tumor.
  • Tumor metastasis typically requires local progression, passive transport, lodgement and proliferation at a remote site. This process also requires the development of tumor vascularization, a process termed angiogenesis. Therefore, by “tumor progression” and “metastasis,” we also include the process of tumor angiogenesis.
  • pre-malignant conditions or "pre-malignant lesion” is meant a cell or tissue which has the potential to turn malignant or metastatic.
  • Pre-malignant lesions include, but are not limited to: atypical ductal hyperplasia of the breast, actinic keratosis (AK), leukoplakia, Barrett's epithelium (columnar metaplasia) of the esophagus, ulcerative colitis, adenomatous colorectal polyps, erythroplasia of Queyrat, Bowen's disease, bowenoid papulosis, vulvar intraepithelial neoplasia (VIN), and displastic changes to the cervix.
  • AK actinic keratosis
  • AK actinic keratosis
  • leukoplakia Barrett's epithelium (columnar metaplasia) of the esophagus
  • ulcerative colitis adenomatous color
  • a “therapeutically effective dose” or “therapeutic dose” is an amount sufficient to effect desired clinical results (i.e., achieve therapeutic efficacy). For example, a
  • therapeutically effective dose or amount of a compound is intended to be an amount that, when the compound is administered as described herein, brings about a positive therapeutic response, such as an amount having anti-tumor activity.
  • a positive therapeutic response may include preventing or delaying progression of carcinoma-in-situ to invasive carcinoma.
  • a therapeutically effective dose can be administered in one or more administrations.
  • a therapeutically effective dose of a compound and/or composition e.g., compositions that include the compound
  • the terms “recipient”, “individual”, “subject”, “host”, and “patient”, are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans.
  • "Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, camels, etc. In some embodiments, the mammal is human. In some cases, the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models, including, but not limited to, rodents including mice, rats, and hamsters; and primates.
  • the terms “specific binding,” “specifically binds,” and the like, refer to non-covalent or covalent preferential binding to a molecule relative to other molecules or moieties in a solution or reaction mixture.
  • the affinity of one molecule for another molecule to which it specifically binds is characterized by a KD (dissociation constant) of 10 " 5 M or less (e.g., 10 "6 M or less, 10 "7 M or less, 10 “8 M or less, 10 “9 M or less, 10 "10 M or less, 10 11 M or less, 10 12 M or less, 10 13 M or less, 10 14 M or less, 10 15 M or less, or 10 16 M or less).
  • KD dissociation constant
  • treatment refers to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.
  • polypeptide and “protein” are used interchangeably throughout the application and mean at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides.
  • a polypeptide may be made up of naturally occurring amino acids and peptide bonds, synthetic peptidomimetic structures, or a mixture thereof.
  • amino acid or “peptide residue”, as used herein encompasses both naturally occurring and synthetic amino acids and includes optical isomers of naturally occurring (genetically encodable) amino acids, as well as analogs thereof. For example, homo-phenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention.
  • Amino acid also includes imino acid residues such as proline and hydroxyproline.
  • the side chains may be in either the D- or the L- configuration. If non- naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradation.
  • amino acid encompasses a- and ⁇ -amino acids.
  • polypeptides may be of any length, e.g., greater than 2 amino acids, greater than 4 amino acids, greater than about 10 amino acids, greater than about 20 amino acids, greater than about 50 amino acids, greater than about 100 amino acids, greater than about 300 amino acids, usually up to about 500 or 1000 or more amino acids.
  • “Peptides” are generally greater than 2 amino acids, greater than 4 amino acids, greater than about 10 amino acids, greater than about 20 amino acids, usually up to about 10, 20, 30, 40 or 50 amino acids.
  • peptides are between, 3 and 5 or 5 and 30 amino acids in length.
  • a peptide may be three or four amino acids in length.
  • fusion protein or grammatical equivalents thereof is meant a protein composed of a plurality of polypeptide components that while typically unjoined in their native state, typically are joined by their respective amino and carboxyl termini through a peptide linkage to form a single continuous polypeptide. Fusion proteins may be a combination of two, three or even four or more different proteins.
  • polypeptide includes fusion proteins, including, but not limited to, a fusion of two or more heterologous amino acid sequences, a fusion of a polypeptide with: a heterologous targeting sequence, a linker, an immunologically tag, a detectable fusion partner, such as a fluorescent protein, ⁇ - galactosidase, luciferase, etc., and the like.
  • deletion is defined as a change in the sequence of a polypeptide in which one or more residues are absent as compared to a sequence of a parental polypeptide.
  • a deletion can remove about 2, about 5, about 10, up to about 20, up to about 30 or up to about 50 or more amino acids.
  • a polypeptide may contain more than one deletion.
  • insertion or “addition” is a change in a sequence of a polypeptide that results in the addition of one or more residues, as compared to a sequence of a parental polypeptide.
  • “Insertion” generally refers to addition to one or more residues within a polypeptide, while “addition” can be an insertion or refer to amino acid residues added at an end, or both termini, of a polypeptide.
  • An insertion or addition is usually of about 1, about 3, about 5, about 10, up to about 20, up to about 30 or up to about 50 or more amino acids.
  • a polypeptide may contain more than one insertion or addition.
  • substitution results from the replacement of one or more residues of a polypeptide by different residues, as compared to a sequence of a parental polypeptide. It is understood that a polypeptide may have conservative amino acid substitutions which, in some case, may have substantially no effect on activity of the polypeptide. By conservative substitutions is intended combinations such as gly, ala; val, ile, leu; asp, glu; asn, gin; ser, thr; lys, arg; and phe, tyr.
  • assessing includes any form of measurement, and includes determining if an element is present or not.
  • determining includes determining if an element is present or not.
  • evaluating means assessing, assessing and “assaying” are used interchangeably and includes quantitative and qualitative determinations. Assessing may be relative or absolute. "Assessing the presence of includes determining the amount of something present, and/or determining whether it is present or absent.
  • non-naturally occurring refers to a composition that does not exist in nature. Any protein described herein may be non-naturally occurring, where the term “non- naturally occurring” refers to a protein that has an amino acid sequence and/or a post- translational modification pattern that is different to the protein in its natural state. For example, a non-naturally occurring protein may have one or more amino acid substitutions, deletions or insertions at the N-terminus, the C-terminus and/or between the N- and C- termini of the protein.
  • a "non-naturally occurring" protein may have an amino acid sequence that is different to a naturally occurring amino acid sequence (i.e., having less than 100% sequence identity to the amino acid sequence of a naturally occurring protein) but that that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to the naturally occurring amino acid sequence.
  • a non-naturally occurring protein may contain an N-terminal methionine or may lack one or more post-translational modifications (e.g., glycosylation, phosphorylation, etc.) if it is produced by a different (e.g., bacterial) cell.
  • a “mutant” or “variant” protein may have one or more amino acid substitutions relative to a wild-type protein and may include a “fusion” protein.
  • the term “fusion protein” refers to a protein composed of a plurality of polypeptide components that are unjoined in their native state. Fusion proteins may be a combination of two, three or even four or more different proteins.
  • polypeptide includes fusion proteins, including, but not limited to, a fusion of two or more heterologous amino acid sequences, a fusion of a polypeptide with: a heterologous targeting sequence, a linker, an immunologically tag, a detectable fusion partner, such as a fluorescent protein, ⁇ - galactosidase, luciferase, etc., and the like.
  • a fusion protein may have one or more heterologous domains added to the N-terminus, C-terminus, and or the middle portion of the protein. If two parts of a fusion protein are "heterologous", they are not part of the same protein in its natural state.
  • non-naturally occurring refers to: a) a combination of components that are not combined by nature, e.g., because they are at different locations, in different cells or different cell compartments; b) a combination of components that have relative concentrations that are not found in nature; c) a combination that lacks something that is usually associated with one of the components in nature; d) a combination that is in a form that is not found in nature, e.g., dried, freeze dried, crystalline, aqueous; and/or e) a combination that contains a component that is not found in nature.
  • a preparation may contain a "non-naturally occurring" buffering agent (e.g., Tris, HEPES, TAPS, MOPS, tricine or MES), a pharmaceutically acceptable carrier (e.g., phosphate buffered saline (PBS), a detergent, a dye, a reaction enhancer or inhibitor, an oxidizing agent, a reducing agent, a solvent or a preservative that is not found in nature.
  • a buffering agent e.g., Tris, HEPES, TAPS, MOPS, tricine or MES
  • a pharmaceutically acceptable carrier e.g., phosphate buffered saline (PBS)
  • PBS phosphate buffered saline
  • detergent e.g., a dye, a reaction enhancer or inhibitor, an oxidizing agent, a reducing agent, a solvent or a preservative that is not found in nature.
  • the term "matriptase protease” refers to the protein having an activity defined by EC 3.4.21.109 and encoded by the human ST14 (suppression of tumorigenicity 14) gene defined by NCBI's Gene ID 6768, and the protein encoded by TMPRSS6, also known as matriptase 2, as well as orthologs from other species (see, e.g., rat, mouse, cow, etc.).
  • Matriptase is a type II transmembrane serine protease expressed in most human epithelia, where it is coexpressed with its cognate transmembrane inhibitor, hepatocyte growth factor activator inhibitor (HAI)-l.
  • Matriptase has an essential physiological role in profilaggrin processing, corneocyte maturation, and lipid matrix formation associated with terminal differentiation of the oral epithelium and the epidermis, and is also critical for hair follicle growth.
  • Matriptase is an 80- to 90-kDa cell surface glycoprotein with a complex modular structure that is common to all matriptases. This protease forms a complex with the Kunitz- type serine protease inhibitor, HAI-1, and is found to be activated by sphingosine-1- phosphate.
  • This protease has been shown to cleave and activate hepatocyte growth factor/scatter factor, and urokinase plasminogen activator.
  • the expression of this protease has been associated with breast, colon, prostate, and ovarian tumors, which implicates its role in cancer invasion, and metastasis.
  • Matriptase and HAI-1 expression are frequently dysregulated in human cancer, and matriptase expression that is unopposed by HAI-1 potently promotes carcinogenesis and metastatic dissemination in animal models.
  • the structure, function and role of matriptase in cancer and other diseases has been extensively reviewed (see, e.g., Uhland Cell. Mol. Life Sci. 2007 63: 2968-78 and Tanabe. FEBS J. 2017 284: 1421-1436).
  • a matriptase substrate refers to a protein cleaved by matriptase.
  • Naturally occurring substrates for matriptase include pro-hepatocyte growth factor (Pro-HGF), urokinase plasminogen activator (uPA), pro-macrophage stimulating protein (Pro-MSP), and platelet derived growth factor-D (PDGF-D), although others are known.
  • Non-naturally occurring substrates for matriptase can be ready designed.
  • Kunitz domain refers to an active domain of a Kunitz-type protease inhibitor. Such a domain typically has a length of about 50 to 60 amino acids. Kunitz domains are stable as standalone peptides, able to recognize specific protein structures, and also work as competitive protease inhibitors in their free form. They have a disulfide rich alpha+beta fold structure. The structures of several Kunitz domains, including the Kunitz domains of HAI-1, have been elucidated (see, e.g., Liu et al, J. Biol. Chem. 2017 292, 8412-8423 and Zhao et al, J. Biol. Chem. 2013 288: 11155-64).
  • HAI-1 refers to a secreted serine-type endopeptidase inhibitor encoded by the human SPINT1 gene, defined by NCBI's Gene ID 6692 and Genbank accession no. AAP36093.1, as well as orthologs thereof. This protein is also known as "Hepatocyte growth factor activator inhibitor-1" and "Kunitz-type protease inhibitor 1". HAI-lhas been extensively studied (see, e.g., Liu et al Acta Crystallogr. Struct. Biol.
  • multimerization domain refers to a domain that can be placed into another protein to make that protein multimerize, e.g., dimerize.
  • nucleic acid includes a plurality of such nucleic acids
  • compound includes reference to one or more compounds and equivalents thereof known to those skilled in the art, and so forth.
  • the practice of the present invention may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and immunology, which are within the skill of the art.
  • Such conventional techniques include polymer array synthesis, hybridization, ligation, and detection of hybridization using a label. Specific illustrations of suitable techniques can be had by reference to the example herein below. However, other equivalent conventional procedures can, of course, also be used.
  • Such conventional techniques and descriptions can be found in standard laboratory manuals such as Genome Analysis: A Laboratory Manual Series (Vols.
  • polypeptide comprising a first Kunitz domain that is at least 90% identical, e.g., at least 95% identical, at least 97% identical, at least 98% identical, at least 99% identical or 100% identical to the entire contiguous length of the KD2/1 fusion of SEQ ID NO: 1 : CVDLPDTGRCRGSFPRWYYDPTEQICKSFVYGGCLGNKNNYLREEECIL ACRGV.
  • the polypeptide may have up to 10 amino acid
  • substitutions e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, relative to SEQ ID NO: l.
  • the amino acid substitutions may be at, e.g., one or more of the following positions of SEQ ID NO: 10: 4, 5, 8, 13-15, 17-25, 27-33, 35, 36, 38, 39, 41-46, 48 and 49, e.g., positions 4, 5, 20-25, 28, 35, 38, 39, 41, 42 and 48.
  • positions 1-3, 6-7, 9-12, 16, 26, 34, 37, 40, 47 and 51-54 should be same as in SEQ ID NO: 1.
  • the first Kunitz domain should bind to and inhibit matriptase.
  • the polypeptide may exhibit high affinity binding to matriptase.
  • the polypeptide may binds to matriptase with an affinity of less than 10 "5 , less than 10 "6 or less than 10 "7 M.
  • Shortening the KD2/1 domain of SEQ ID NO: 1 may cause the protein to have reduced function.
  • the reason for this is due to the cysteine residues at positions 250 and 300 (Fig. 13), essentially the book ends of the KD2/1 sequence. From the KDl -matriptase crystal structure information, it is clear that these residues form a disulfide bond, potentially important for overall structure formation and orientation of the matriptase binding motif. Ablation of these critical residues would therefore may be detrimental for the overall function of the KD2/1 domain.
  • residues flanking the C-terminal cysteine are likely critical for the formation of the alpha helix secondary structure and likely important, possibly necessary for proper overall fold, flexibility, and function of the KD2/1 domain. This is particularly relevant once KD2/1 is expressed within the context of the complete HAI-1 protein, as we observed when comparing soluble expression potential of KDlx2-Fc with KD1-KD2/1-Fc; in which KDlx2-Fc had extremely poor expression due to improper folded domains (Fig. 7). Thus, any attempts to further minimize the functional region of KD2/1 may result in deleterious effects to the overall inhibitor structure and function.
  • KDl-matriptase crystal structure as a surrogate for mutational analysis (Fig. 13), it appears that the KD2/1 domain may be further mutagenized at some regions and not lose complete function.
  • known areas that may likely will result in loss of function if mutated include: the primary binding motif (R258, C259, R260, G261); the secondary binding regions (R265, N286, N289); and cysteine residues
  • Regions that will be less likely to result in loss of function with mutations include: the KD2 region of the KD2/1 chimera (V251 to T256), the secondary protein structures (F263 to Y268; K276 to Y280; R292 to R301), and the glycine (G257- 281-282-285-302) and serine (S262) residues throughout the KD2/1 sequence. These regions are believed to be important for overall fold, flexibility, and orientation of the secondary structures, primary and secondary binding motif, and formation of the disulfide bonds;
  • the polypeptide may further comprise a second Kunitz domain, e.g., another Kunitz domain that is capable of inhibiting matriptase.
  • the second Kunitz domain may have an amino acid sequence that is at least 90% identical to, e.g., at least 95% identical to, at least 97% identical to, at least 98% identical, at least 99% identical to or 100% identical to the entire contiguous length of the Kunitz domain of SEQ ID NO:2: CLASNKVGRCRGSFPRWYYDPTEQICKSFVY
  • GGCLGN KNNYLREEECILACRGV Other Kunitz domains that inhibit matriptase are known and can be used.
  • the polypeptide may be comprised within a larger protein which, in some embodiments, may comprise a sequence that is at least 90% identical to, e.g., at least 90% sequence identity, at least 95% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to a naturally occurring (e.g., human) Hepatocyte growth factor activator inhibitor-1 (HAI-1).
  • HAI-1 Hepatocyte growth factor activator inhibitor-1
  • use of a human sequence may be less immunogenic as a therapeutic.
  • the polypeptide further comprises multimerization domain, e.g., an Fc domain.
  • An Fc domain increases the valency of the Kunitz domains in the protein and increases the half-life of the protein in the blood.
  • the polypeptide may comprise multiple copies (e.g., 2, 3, 4 or 5 or more copies) of the first Kunitz domain, which may be the same or different to one another.
  • sequence of an example of a present polypeptide is set forth below as SEQ NO: 3, where the sequence in bold is HAI-1, the underlined sequence in bold is the chimeric
  • KD2/1 domain as described above, and the italicized sequence is a human IgGl Fc domain.
  • the polypeptide may contain a sequence that is the KD2/1 domain as shown above, and the italicized sequence is an Fc domain.
  • the polypeptide may contain a first region that has an amino acid sequence that is at least 90% identical to, e.g., at least 95% identical, at least 97% identical, at least
  • amino acids 1-339 of SEQ ID NO: 3 a second region that is at least 90% identical, e.g., at least 95% identical, at least 97% identical, at least 98% identical, at least 99% identical or 100% identical to amino acids 340- 391 of sequence ID NO: 3 (which corresponds to SEQ ID NO: l), a third region that has an amino acid sequence that is at least 90% identical to, e.g., at least 95% identical, at least 97% identical, at least 98% identical, at least 99% identical or 100% identical to amino acids 392- 414 of SEQ ID NO: 3, as well as an Fc domain (e.g., a human Fc domain) at the C-terminus.
  • an Fc domain e.g., a human Fc domain
  • This polypeptide may optionally contain a linker between the between the end of the HAI-1 - based sequence (residue 414) and the beginning of the Fc sequence for ease of cloning and flexibility.
  • the linker may be a flexible linker composed of three or more Gly and/or Ser residues.
  • the polypeptide may be combined with a pharmaceutically acceptable excipient.
  • a pharmaceutically acceptable excipient examples include sodium EDTA
  • a method for inhibiting a matriptase protease is also provided.
  • the method may comprise contacting the matriptase protease with a polypeptide, thereby inhibiting the matriptase protease.
  • the present polypeptide may reduce the protease activity of matriptase by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the protease activity in the absence of the antibody.
  • the contacting may be done in the presence of a matriptase substrate, e.g., pro- hepatocyte growth factor (Pro-HGF), urokinase plasminogen activator (uPA), pro- macrophage stimulating protein (Pro-MSP), or platelet derived growth factor-D (PDGF-D).
  • a matriptase substrate e.g., pro- hepatocyte growth factor (Pro-HGF), urokinase plasminogen activator (uPA), pro- macrophage stimulating protein (Pro-MSP), or platelet derived growth factor-D (PDGF-D).
  • the matriptase protease is tethered to the surface of a cell, e.g., a mammalian cell.
  • the fusion protein may comprise, in order, an N-terminal domain of a reporter protein, a cleavage site for a protease (i.e., a sequence- specific protease); and a C- terminal domain of the reporter protein.
  • the cleavage site may be the cleavage site for a mammalian, bacterial or viral protease, e.g., a therapeutic target such as matriptase or the HIV protease.
  • fusion protein emits an optically- detectable signal (e.g., luminescence or fluorescence) when it is in its uncleaved form.
  • the reporter protein is a fluorescent protein and the optically detectable signal is fluorescence.
  • the reporter protein should be composed of two self- complementing domains that can be split into different (inactive) proteins that can reconstitute the reporter protein when they are brought together.
  • Split reporter proteins which have typically been used to examine inter-molecular interactions (e.g., protein-protein interactions), are described in a variety of publications including, but not limited to Demidov et al (Proc. Natl. Acad. Sci. U S A. 2006 103:2052-6) and Kamiyama et al (Nat. Commun. 2016 7: 11046) and can be readily adapted for use herein.
  • the cleavage site may have the amino acid sequence of any of the cleavage sites described herein, e.g., the B4 amino acid sequence.
  • the N- and C-terminal domains of the fustion protein do not contain any cleavage sites for the protease.
  • a method comprising (a) contacting the fusion protein with the protease and a candidate inhibitor for the protease; and (b) measuring the optically- detectable signal, e.g., fluorescence.
  • the candidate inhibitor may be proteinaceous and, in some cases, may be a naturally occurring polypeptide or a non- naturally occurring variant of the same (e.g., HAI-1 or a variant of the same).
  • the candidate inhibitor may be expressed by a cell (e.g., a yeast cell) such that the cell either secretes the candidate inhibitor or presents the candidate inhibitor on its surface.
  • the method may comprise contacting the fusion protein with a cell, wherein the cell either secretes the candidate inhibitor or presents the candidate inhibitor on its surface.
  • the method may be used to screen for protease inhibitors.
  • the method may comprise contacting the fusion protein with a plurality of cells, wherein the cells express multiple candidate inhibitors (i.e., they contain a construct that encodes the candidate inhibitors) and measuring the optically-detectable signal for each candidate inhibitor.
  • the cells are yeast cells that display the candidate inhibitors, although other cell types can be used.
  • This method may comprise administering a therapeutically effective amount of the polypeptide to the subject.
  • the matriptase-related disease or condition is cancer, iron overload disease, osteoarthritis, influenza or human immunodeficiency virus.
  • Acute Lymphoblastic Leukemia ALL
  • Acute Myeloid Leukemia AML
  • Adrenocortical Carcinoma AIDS-Related Cancers
  • Anal Cancer Appendix Cancer
  • Astrocytomas Atypical Teratoid/Rhabdoid Tumor
  • Basal Cell Carcinoma Basal Cell Carcinoma
  • Bile Duct Cancer Extrahepatic
  • Bladder Cancer Bone Cancer (e.g., Ewing Sarcoma, Osteosarcoma and Malignant Fibrous Histiocytoma, etc.), Brain Stem Glioma, Brain Tumors (e.g., Astrocytomas, Central Nervous System Embryonal Tumors, Central Nervous System Germ Cell Tumors, Craniophary
  • Lymphoma Cervical Cancer, Childhood Cancers, Chordoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), Chronic Myeloproliferative Neoplasms, Colon Cancer, Colorectal Cancer, Craniopharyngioma, Cutaneous T-Cell Lymphoma, Duct (e.g., Bile Duct, Extrahepatic, etc.), Ductal Carcinoma In situ (DCIS), Embryonal Tumors, Endometrial Cancer, Ependymoma, Esophageal Cancer,
  • CLL Chronic Lymphocytic Leukemia
  • CML Chronic Myelogenous Leukemia
  • Neoplasms Colon Cancer
  • Colorectal Cancer Craniopharyngioma
  • Duct e.g., Bile Duct, Extrahepatic, etc.
  • DCIS Ductal Carcinoma In Situ
  • Embryonal Tumors Endometrial Cancer
  • Esthesioneuroblastoma Ewing Sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer (e.g., Intraocular Melanoma, Retinoblastoma, etc.), Fibrous Histiocytoma of Bone (e.g., Malignant, Osteosarcoma, ect.), Gallbladder Cancer, Gastric (Stomach) Cancer, Gastrointestinal Carcinoid Tumor,
  • GIST Gastrointestinal Stromal Tumors
  • Germ Cell Tumor e.g., Extracranial
  • Extragonadal, Ovarian, Testicular, etc. Gestational Trophoblastic Disease, Glioma, Hairy Cell Leukemia, Head and Neck Cancer, Heart Cancer, Hepatocellular (Liver) Cancer, Histiocytosis (e.g., Langerhans Cell, etc.), Hodgkin Lymphoma, Hypopharyngeal Cancer, Intraocular Melanoma, Islet Cell Tumors (e.g., Pancreatic Neuroendocrine Tumors, etc.), Kaposi Sarcoma, Kidney Cancer (e.g., Renal Cell, Wilms Tumor, Childhood Kidney Tumors, etc.), Langerhans Cell Histiocytosis, Laryngeal Cancer, Leukemia (e.g., Acute Lymphoblastic (ALL), Acute Myeloid (AML), Chronic Lymphocytic (CLL), Chronic Myelogenous (CML), Hairy Cell, etc.), Lip and Oral Cavity Cancer, Liver Cancer (Prim
  • Rhabdomyosarcoma Salivary Gland Cancer
  • Sarcoma e.g., Ewing, Kaposi, Osteosarcoma, Rhabdomyosarcoma, Soft Tissue, Uterine, etc.
  • Sezary Syndrome Skin Cancer (e.g., Childhood, Melanoma, Merkel Cell Carcinoma, Nonmelanoma, etc.), Small Cell Lung
  • the cancer to be treated is an epithelial cancer (i.e., a carcinoma) or a cancer derived from an epithelial cell type, including but not limited to, e.g.: acinar carcinoma , acinic cell carcinoma, acinous carcinoma, adenocystic carcinoma , adenoid cystic carcinoma, adenosquamous carcinoma, adnexal carcinoma, adrenocortical carcinoma, alveolar carcinoma, ameloblastic carcinoma, apocrine carcinoma, basal cell carcinoma,
  • bronchioloalveolar carcinoma bronchogenic carcinoma, cholangiocellular carcinoma, chorionic carcinoma, clear cell carcinoma, colloid carcinoma, cribriform carcinoma, ductal carcinoma in situ, embryonal carcinoma, carcinoma en cuirasse, endometrioid carcinoma, epidermoid carcinoma, carcinoma ex mixed tumor, carcinoma ex pleomorphic adenoma, follicular carcinoma of thyroid gland, hepatocellular carcinoma, carcinoma in situ, intraductal carcinoma, Hurthle cell carcinoma, inflammatory carcinoma of the breast, large cell carcinoma, invasive lobular carcinoma, lobular carcinoma, lobular carcinoma in situ (LCIS), medullary carcinoma, meningeal carcinoma, Merkel cell carcinoma, mucinous carcinoma, mucoepidermoid carcinoma, nasopharyngeal carcinoma, non-small cell carcinoma , non-small cell lung carcinoma (NSCLC), oat cell carcinoma, papillary carcinoma, renal cell carcinoma, scirrhous carcinoma, sebaceous carcinoma, carcinoma simplex, signet-ring cell carcinoma,
  • the polypeptide may inhibit cancer progression, e.g., cancer metastasis, in the subject.
  • a subject polypeptide in the subject methods, can be administered to the host using any convenient means capable of resulting in the desired therapeutic effect or diagnostic effect.
  • the agent can be incorporated into a variety of formulations for therapeutic administration. More particularly, a subject polypeptide can be formulated into
  • compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.
  • a subject polypeptide in pharmaceutical dosage forms, can be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds.
  • the following methods and excipients are merely exemplary and are in no way limiting.
  • a subject polypeptide can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
  • conventional additives such as lactose, mannitol, corn starch or potato starch
  • binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins
  • disintegrators such as corn starch, potato starch or sodium carboxymethylcellulose
  • lubricants such as talc or magnesium stearate
  • a subject polypeptide can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • Pharmaceutical compositions comprising a subject polypeptide are prepared by mixing the polypeptide having the desired degree of purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers and/or tonicity agents.
  • Acceptable carriers, excipients and/or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m- cresol, methyl or propyl parabens, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as ge
  • the pharmaceutical composition may be in a liquid form, a lyophilized form or a liquid form reconstituted from a lyophilized form, wherein the lyophilized preparation is to be reconstituted with a sterile solution prior to administration.
  • the standard procedure for reconstituting a lyophilized composition is to add back a volume of pure water (typically equivalent to the volume removed during lyophilization); however solutions comprising antibacterial agents may be used for the production of pharmaceutical compositions for parenteral administration; see also Chen (1992) Drug Dev Ind Pharm 18, 1311-54.
  • Exemplary polypeptide concentrations in a subject pharmaceutical composition may range from about 1 mg/mL to about 200 mg/ml or from about 50 mg/mL to about 200 mg/mL, or from about 150 mg/mL to about 200 mg/mL.
  • An aqueous formulation of the polypeptide may be prepared in a pH-buffered solution, e.g., at pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5.
  • buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers.
  • the buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation.
  • a tonicity agent may be included in the polypeptide formulation to modulate the tonicity of the formulation.
  • Exemplary tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof.
  • the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable.
  • isotonic denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum.
  • Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 nM.
  • a surfactant may also be added to the polypeptide formulation to reduce aggregation of the formulated polypeptide and/or minimize the formation of particulates in the formulation and/or reduce adsorption.
  • exemplary surfactants include polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS).
  • polyoxyethylenesorbitan-fatty acid esters examples include polysorbate 20, (sold under the trademark Tween 20TM) and polysorbate 80 (sold under the trademark Tween 80TM).
  • suitable polyethylene-polypropylene copolymers examples include those sold under the names Pluronic® F68 or Poloxamer 188TM.
  • suitable Polyoxyethylene alkyl ethers are those sold under the trademark BrijTM.
  • Exemplary concentrations of surfactant may range from about 0.001% to about 1% w/v.
  • a lyoprotectant may also be added in order to protect the labile active ingredient (e.g. a protein) against destabilizing conditions during the lyophilization process.
  • lyoprotectants include sugars (including glucose and sucrose); polyols (including mannitol, sorbitol and glycerol); and amino acids (including alanine, glycine and glutamic acid). Lyoprotectants can be included in an amount of about 10 mM to 500 nM.
  • a subject formulation includes a subject polypeptide, and one or more of the above-identified agents (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof.
  • a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% (w/v).
  • a subject formulation can be a liquid or lyophilized formulation suitable for parenteral administration, and can comprise: about 1 mg/mL to about 200 mg/mL of a subject polypeptide; about 0.001 % to about 1 % of at least one surfactant; about 1 mM to about 100 mM of a buffer; optionally about 10 mM to about 500 mM of a stabilizer; and about 5 mM to about 305 mM of a tonicity agent; and has a pH of about 4.0 to about 7.0.
  • a subject parenteral formulation is a liquid or lyophilized formulation comprising: about 1 mg/mL to about 200 mg/mL of a subject polypeptide; 0.04% Tween 20 w/v; 20 mM L-histidine; and 250 mM Sucrose; and has a pH of 5.5.
  • a subject parenteral formulation comprises a lyophilized formulation comprising: 1) 15 mg/mL of a subject polypeptide; 0.04% Tween 20 w/v; 20 mM L-histidine; and 250 mM sucrose; and has a pH of 5.5; or 2) 75 mg/mL of a subject polypeptide; 0.04% Tween 20 w/v; 20 mM L-histidine; and 250 mM sucrose; and has a pH of 5.5;or 3) 75 mg/mL of a subject polypeptide; 0.02% Tween 20 w/v; 20 mM L-histidine; and 250 mM Sucrose; and has a pH of 5.5; or 4) 75 mg/mL of a subject polypeptide; 0.04% Tween 20 w/v; 20 mM L-histidine; and 250 mM trehalose; and has a pH of 5.5; or 6) 75 mg/mL of a subject polypeptide;
  • a subject parenteral formulation is a liquid formulation comprising: 1) 7.5 mg/mL of a subject polypeptide; 0.022% Tween 20 w/v; 120 mM L- histidine; and 250 125 mM sucrose; and has a pH of 5.5; or 2) 37.5 mg/mL of a subject polypeptide; 0.02% Tween 20 w/v; 10 mM L-histidine; and 125 mM sucrose; and has a pH of 5.5; or 3) 37.5 mg/mL of a subject polypeptide; 0.01% Tween 20 w/v; 10 mM L-histidine; and 125 mM sucrose; and has a pH of 5.5; or 4) 37.5 mg/mL of a subject polypeptide; 0.02% Tween 20 w/v; 10 mM L-histidine; 125 mM trehalose; and has a pH of 5.5; or 5) 37.5 mg/mL of a
  • a subject polypeptide can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases.
  • bases such as emulsifying bases or water-soluble bases.
  • a subject polypeptide can be administered rectally via a suppository.
  • the suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
  • Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more inhibitors.
  • unit dosage forms for injection or intravenous administration may comprise a subject polypeptide in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.
  • the specifications for a subject polypeptide may depend on the particular polypeptide employed and the effect to be achieved, and the pharmacodynamics associated with each polypeptide in the host.
  • a subject polypeptide can be formulated in suppositories and, in some cases, aerosol and intranasal compositions.
  • the vehicle composition will include traditional binders and carriers such as, polyalkylene glycols, or triglycerides.
  • suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10% (w/w), e.g., about 1% to about 2%.
  • Intranasal formulations will usually include vehicles that neither cause irritation to the nasal mucosa nor significantly disturb ciliary function.
  • Diluents such as water, aqueous saline or other known substances can be employed with the subject invention.
  • the nasal formulations may also contain preservatives such as, but not limited to, chlorobutanol and benzalkonium chloride.
  • a surfactant may be present to enhance absorption of the subject proteins by the nasal mucosa.
  • a subject polypeptide can be administered as an injectable formulation.
  • injectable compositions are prepared as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or the polypeptide encapsulated in liposome vehicles.
  • Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof.
  • the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents.
  • auxiliary substances such as wetting or emulsifying agents or pH buffering agents.
  • Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985.
  • the composition or formulation to be administered will, in any event, contain a quantity of a subject polypeptide adequate to achieve the desired state in the subject being treated.
  • the pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers or diluents, are readily available to the public.
  • pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
  • a subject polypeptide is formulated in a controlled release formulation.
  • Sustained-release preparations may be prepared using methods well known in the art. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide in which the matrices are in the form of shaped articles, e.g. films or microcapsules. Examples of sustained-release matrices include polyesters, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, hydrogels, polylactides, degradable lactic acid-glycolic acid copolymers and poly-D-(-)-3-hydroxybutyric acid. Possible loss of biological activity and possible changes in immunogenicity of antibodies comprised in sustained-release preparations may be prevented by using appropriate additives, by controlling moisture content and by developing specific polymer matrix compositions.
  • Controlled release within the scope of this invention can be taken to mean any one of a number of extended release dosage forms.
  • the following terms may be considered to be substantially equivalent to controlled release, for the purposes of the present invention: continuous release, controlled release, delayed release, depot, gradual release, long-term release, programmed release, prolonged release, proportionate release, protracted release, repository, retard, slow release, spaced release, sustained release, time coat, timed release, delayed action, extended action, layered-time action, long acting, prolonged action, repeated action, slowing acting, sustained action, sustained-action medications, and extended release. Further discussions of these terms may be found in Lesczek Krowczynski, Extended- Release Dosage Forms. 1987 (CRC Press, Inc.).
  • Controlled release technologies cover a very broad spectrum of drug dosage forms. Controlled release technologies include, but are not limited to physical systems and chemical systems.
  • Physical systems include, but are not limited to, reservoir systems with rate- controlling membranes, such as microencapsulation, macroencapsulation, and membrane systems; reservoir systems without rate-controlling membranes, such as hollow fibers, ultra microporous cellulose triacetate, and porous polymeric substrates and foams; monolithic systems, including those systems physically dissolved in non-porous, polymeric, or elastomeric matrices (e.g., nonerodible, erodible, environmental agent ingression, and degradable), and materials physically dispersed in non-porous, polymeric, or elastomeric matrices (e.g., nonerodible, erodible, environmental agent ingression, and degradable); laminated structures, including reservoir layers chemically similar or dissimilar to outer control layers; and other physical methods, such as osmotic pumps, or adsorption onto ion- exchange resins.
  • rate- controlling membranes such as microencapsulation, macroencapsulation, and membrane systems
  • Chemical systems include, but are not limited to, chemical erosion of polymer matrices (e.g., heterogeneous, or homogeneous erosion), or biological erosion of a polymer matrix (e.g., heterogeneous, or homogeneous). Additional discussion of categories of systems for controlled release may be found in Agis F. Kydonieus, Controlled Release Technologies: Methods, Theory and Applications, 1980 (CRC Press, Inc.).
  • controlled release drug formulations that are developed for oral administration. These include, but are not limited to, osmotic pressure-controlled gastrointestinal delivery systems; hydrodynamic pressure-controlled gastrointestinal delivery systems; membrane permeation-controlled gastrointestinal delivery systems, which include microporous membrane permeation-controlled gastrointestinal delivery devices; gastric fluid-resistant intestine targeted controlled-release gastrointestinal delivery devices; gel diffusion-controlled gastrointestinal delivery systems; and ion-exchange-controlled gastrointestinal delivery systems, which include cationic and anionic drugs. Additional information regarding controlled release drug delivery systems may be found in Yie W. Chien, Novel Drug Delivery Systems, 1992 (Marcel Dekker, Inc.).
  • a suitable dosage can be determined by an attending physician or other qualified medical personnel, based on various clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex of the patient, time, and route of administration, general health, and other drugs being administered concurrently.
  • a subject polypeptide may be administered in amounts between 1 ng/kg body weight and 20 mg/kg body weight per dose, e.g. between 0.1 mg/kg body weight to 10 mg/kg body weight, e.g. between 0.5 mg/kg body weight to 5 mg/kg body weight; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. If the regimen is a continuous infusion, it can also be in the range of 1 ⁇ g to 10 mg per kilogram of body weight per minute.
  • dose levels can vary as a function of the specific polypeptide, the severity of the symptoms and the susceptibility of the subject to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.
  • a subject polypeptide is administered to an individual using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and localized routes of administration.
  • a subject polypeptide composition can be administered in a single dose or in multiple doses. In some embodiments, a subject polypeptide composition is administered orally. In some embodiments, a subject polypeptide composition is administered via an inhalational route. In some embodiments, a subject polypeptide composition is administered intranasally. In some embodiments, a subject polypeptide composition is administered locally. In some embodiments, a subject polypeptide composition is administered intracranially. In some embodiments, a subject polypeptide composition is administered intravenously.
  • the agent can be administered to a host using any available conventional methods and routes suitable for delivery of conventional drugs, including systemic or localized routes.
  • routes of administration contemplated by the invention include, but are not necessarily limited to, enteral, parenteral, or inhalational routes.
  • Parenteral routes of administration other than inhalation administration include, but are not necessarily limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, and intravenous routes, i.e. , any route of
  • parenteral administration can be carried to effect systemic or local delivery of a subject polypeptide. Where systemic delivery is desired, administration typically involves invasive or systemically absorbed topical or mucosal administration of pharmaceutical preparations.
  • a subject polypeptide can also be delivered to the subject by enteral administration.
  • Enteral routes of administration include, but are not necessarily limited to, oral and rectal (e.g. , using a suppository) delivery.
  • treatment is meant at least an amelioration of the symptoms associated with the pathological condition afflicting the host, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. symptom, associated with the pathological condition being treated, such as muscle atrophy.
  • amelioration also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g. prevented from happening, or stopped, e.g.
  • a subject polypeptide is administered by injection and/or delivery, e.g., to a site in a brain artery or directly into brain tissue.
  • a subject antibody can also be administered directly to a target site e.g., by biolistic delivery to the target site.
  • hosts are treatable according to the subject methods.
  • hosts are “mammals” or “mammalian,” where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (e.g. , dogs and cats), rodentia (e.g. , mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys).
  • the hosts will be humans.
  • Kits with unit doses of a subject antibody e.g. in oral or injectable doses, are provided.
  • kits in addition to the containers containing the unit doses will be an informational package insert describing the use and attendant benefits of the antibody in treating pathological condition of interest.
  • Preferred compounds and unit doses are those described herein above.
  • Embodiment 1 A polypeptide comprising a first Kunitz domain that is at least 90% identical to the entire contiguous length of the KD2/1 fusion of SEQ ID NO: 1.
  • Embodiment 2 The polypeptide of embodiment 1, wherein the polypeptide further comprises a second Kunitz domain.
  • Embodiment 3 The polypeptide of any prior embodiment, wherein the second Kunitz domain has an amino acid sequence that is at least 90% identical to the entire contiguous length of the Kunitz domain of SEQ ID NO:2.
  • Embodiment 4 The polypeptide of any prior embodiment, wherein the polypeptide comprises a sequence that is at least 90% identical to human Hepatocyte growth factor activator inhibitor- 1 (HAI-1).
  • HAI-1 human Hepatocyte growth factor activator inhibitor-1
  • Embodiment 5 The polypeptide of any prior embodiment, wherein the polypeptide further comprises multimerization domain.
  • Embodiment 6 The polypeptide of any prior embodiment, wherein the polypeptide comprises an Fc domain.
  • Embodiment 7 The polypeptide of any prior embodiment, wherein the first Kunitz domain of the polypeptide is at least 95% identical to the entire contiguous length of SEQ ID NO: 1.
  • Embodiment 8A The polypeptide of any prior embodiment, wherein the first Kunitz domain of the polypeptide is identical to the entire contiguous length of SEQ ID NO: 1.
  • Embodiment 8B The polypeptide of any prior embodiment, wherein the polypeptide may contain a first region that has an amino acid sequence that is at least 90% identical to amino acids 1-339 of SEQ ID NO: 3, a second region that is at least 90% identical to amino acids 340-391 of sequence ID NO: 3, and a third region that has an amino acid sequence that is at least 90% identical to amino acids 392-414 of SEQ ID NO: 3, as well as a C- terminal Fc domain.
  • Embodiment 8C The polypeptide of any prior embodiment, wherein the polypeptide may contain a first region that has an amino acid sequence that is at least 95% identical to amino acids 1-339 of SEQ ID NO: 3, a second region that is at least 95% identical to amino acids 340-391 of sequence ID NO: 3, and a third region that has an amino acid sequence that is at least 95% identical to amino acids 392-414 of SEQ ID NO: 3, as well as a C- terminal Fc domain.
  • Embodiment 9 The polypeptide of any prior embodiment, wherein the polypeptide comprises multiple copies of the first Kunitz domain.
  • Embodiment 10 A pharmaceutical composition comprising a polypeptide of any prior embodiment and a pharmaceutically acceptable carrier.
  • Embodiment 11 A method for inhibiting a matriptase protease comprising:
  • Embodiment 12 The method of embodiment 11, wherein the matriptase protease is tethered to the surface of a cell.
  • Embodiment 13 The method of any of embodiment 11-12, wherein the contacting is done in the presence of a matriptase substrate.
  • Embodiment 14 The method of embodiment 13, wherein the matriptase substrate is pro-hepatocyte growth factor (Pro-HGF), urokinase plasminogen activator (uPA), pro- macrophage stimulating protein (Pro-MSP), or platelet derived growth factor-D (PDGF-D).
  • Pro-HGF pro-hepatocyte growth factor
  • uPA urokinase plasminogen activator
  • Pro-MSP pro- macrophage stimulating protein
  • PDGF-D platelet derived growth factor-D
  • Embodiment 15 A method of treating a matriptase-related disease or condition in a subject, comprising administering a therapeutically effective amount of the polypeptide of any of embodiments 1-9 to the subject.
  • Embodiment 16 The method of embodiment 15, wherein the matriptase-related disease or condition is cancer, iron overload disease, osteoarthritis, influenza or human immunodeficiency virus.
  • Embodiment 17 The method of any of embodiments 15-16, wherein the polypeptide inhibits cancer progression in the subject.
  • Embodiment 18 The method of any of embodiments 15-17, wherein the cancer is breast, colorectal, pancreatic, cervical, or prostate cancer.
  • Embodiment 19 The method of any of claims 15-18, wherein the polypeptide inhibits metastasis of a cancer in the subject.
  • Embodiment 21 A fusion protein comprising, in order:
  • Embodiment 22 The fusion protein of embodiment 21, wherein reporter protein is a fluorescent protein and the optically detectable signal is fluorescence Embodiment 23.
  • Embodiment 24 The fusion partner of any of embodiments 21-22, wherein the cleavage site has the B4 amino acid sequence.
  • Embodiment 25 A method comprising: (a) contacting a fusion protein of embodiment 1 with a candidate inhibitor for the protease; and (b) measuring the optically- detectable signal.
  • Embodiment 26 The method of embodiment 25, wherein method comprises contacting the fusion protein with a cell, wherein the cell either secretes the candidate inhibitor or presents the candidate inhibitor on its surface.
  • Embodiment 27 The method of embodiments 25 or 26, wherein the candidate agent is a variant of HAI-1.
  • Embodiment 28 The method of any of embodiments 25-27, wherein the method comprises: contacting the fusion protein with a plurality of cells, wherein the cells express different candidate inhibitors; and measuring the optically-detectable signal for each candidate inhibitor.
  • Embodiment 29 The method of embodiment 28, wherein the cells are yeast cells that display the candidate inhibitors.
  • the inactive KD2 domain of HAI- 1 was replaced with an engineered chimeric variant of KD2/KD1 domains, and fused the resulting construct to an antibody Fc domain to increase valency and circulating serum half-life.
  • the engineered inhibitor demonstrates a protease selectivity profile similar to wild-type KDl but distinct from HAI-1, and inhibits activation of pro-HGF, the natural substrate, and matriptase expressed on cancer cells with at least an order of magnitude greater efficacy than KDl.
  • Yeast cell surface binding assays and library screening conditions Induced EBY100 yeast cells were counted, washed, and then mixed with soluble matriptase (final concentration 0 - InM, serial dilution) and matriptase assay buffer at appropriate volumes to account for ligand depletion. Reactions were incubated for 48 hr at room temperature to reach equilibrium. Samples were then incubated with a 1 :250 dilution of anti-HA, mouse primary antibody (Fisher Scientific)) for final 30 min at room temperature.
  • soluble matriptase final concentration 0 - InM, serial dilution
  • matriptase assay buffer at appropriate volumes to account for ligand depletion. Reactions were incubated for 48 hr at room temperature to reach equilibrium. Samples were then incubated with a 1 :250 dilution of anti-HA, mouse primary antibody (Fisher Scientific)) for final 30 min at room temperature.
  • samples were labeled with secondary antibodies to measure yeast expression (1 :100 dilution of anti-mouse phycoerythrin PE (Invitrogen)) and matriptase binding (1: 100 dilution of anti-His fluorescein isothiocyanate FITC (Bethyl)).
  • Samples were incubated at 4°C for 15 min, washed, and maintained on ice until loading onto a flow cytometer for analysis (BD Accuri) or library sorting (BD Aria II). Data analysis and three parameter curve fits performed with GraphPad Prism software, version 6. Sorted cells were recovered in SD- CAA liquid media and incubated at 30°C overnight or until reaching an optical density (O.D.) of 4-8.
  • Yeast surface protein expression was then induced by culturing cells in SG- CAA media at 20°C overnight.
  • the initial library (approximately 5x10 7 yeast) was screened twice in the presence of 10 nM matriptase, then isolated yeast were lysed for DNA extraction (Zymoprep; Fisher Scientific). This DNA was subjected to additional mutagenesis (see above) before retransformation into yeast.
  • This library was screened seven times under equilibrium sorting conditions with 10 nM matriptase. Parallel screening for binding against secondary reagents alone was used to reduce false positives.
  • the final pool of isolated yeast was lysed for DNA extraction (Zymoprep; Fisher Scientific), and transformed into DH10B electrocompetent E. coli cells for plasmid amplification and sequencing (Sequetech, MCLAB).
  • Protease inhibition assay First, 0.05 nM matriptase (R&D Systems) was added to soluble inhibitor (final concentrations ranging from 0-50nM) containing matriptase assay buffer. Soluble matriptase substrate (1 ⁇ ; Boc-QAR-AMC) (R&D Systems) was added to initiate the reaction. Matriptase inhibition assays were carried out on yeast surface displayed proteins using the same matriptase and substrate conditions, except yeast were first counted (ranging from 10 - 10 8 yeast cells/sample) and incubated with matriptase prior to addition of the substrate.
  • Additional protease inhibition assays were carried out using 0.5 nM of urokinase, trypsin 3, kallikrein 4, and hepsin, with 1 ⁇ of each enzyme specific substrate, Z-GGR-AMC, Mca-RPKPVE-Nval-WRK(Dnp)-NH2, Boc-VPR-AMC, and Boc-QRR- AMC, respectively. All enzymes and substrates purchased from R&D Systems were assumed 100% active; buffers and assay conditions were prepared as each R&D Systems protocol describes.
  • Soluble inhibitor (final concentrations ranging from 0-150nM, excluding inhibitor depleting conditions) was added initially to each enzyme containing its respective assay buffer, then the reaction was initiated by addition of substrate and measuring fluorescent output over time at 380nm/460nm (matriptase, urokinase, kallikrein 4, and hepsin) and 320nm/405nm (trypsin 3).
  • Protease activation of fluorescent substrate, relative fluorescent units (RFU)/s was measured for at least 30 min using a kinetic microplate reader (Synergy H4, Biotek), corrected for background, and then converted to initial relative velocity, v/v 0 .
  • Yeast media (YPD media) 20 g/L glucose, 20 g/L peptone, and 10 g/L yeast extract; (SD-CAA media) 20 g/L dextrose, 6.7 g/L yeast nitrogen base lacking amino acids, 5.4 g/L Na2HP04, 8.6 g/L NaH2P04 ⁇ H20, and 5 g/L Bacto casamino acids; (SG-CAA media) same as SD-CAA, but dextrose is substituted for 20 g/L galactose; (Low pH, SD-CAA media) same as to SD-CAA, but phosphate components were substituted with 13.7 g/L sodium citrate dehydrate and 8.4 g/L citric acid anhydrous, and pH was adjusted to 4.5.
  • YPD media 20 g/L glucose, 20 g/L peptone, and 10 g/L yeast extract
  • SD-CAA media 20 g/L dextrose, 6.7 g/L yeast nitrogen
  • BMMY, BMGY media, and RDB plates for P. pastoris strain GS115 were prepared as described (52).
  • Mammalian cells and media (Complete growth media) Dulbecco's modified Eagle medium (Fisher Scientific) containing 10% fetal bovine serum (FBS)(Fisher Scientific).
  • Cell lines used include: Human Embryonic Kidney (HEK) cells, PC3 (prostate cancer), MDA-MB-231 (breast cancer), and A549 (lung cancer) - (ATCC).
  • KD2 variant cloning and library construction The natural KD2 domain is comprised of amino acids Cys375 to Cys425 (Genbank Accession ID: AY358969.1). KD2 variant constructs Arg-Cys-Arg (graft 1), or Arg-Cys-Arg-Gly (graft 2) were created using PCR and gene products were cloned into the pTMY yeast display vector previously described (52) using Nhel and Mlul restriction sites. Plasmids were transformed into the yeast strain EBY100 using electroporation, expanded in SD-CAA media at 30°C, and then induced for surface expression using SG-CAA media at 20°C (45). All yeast displayed proteins were cloned and prepared in this manner.
  • Yeast expression levels were measured using an anti-HA epitope tag antibody (1:250 dilution of anti-HA, mouse primary antibody (Fisher Scientific)), and an anti-HAI-1 antibody (1 : 100 dilution of rabbit anti-HAI-1 primary antibody (Fisher Scientific)), followed by analysis using flow cytometry (BD Accuri).
  • the KD2 graft 2 library was constructed using error-prone PCR as previously reported (46). In short, variable concentrations of Mn 2+ (0.075 mM or 0.15 mM) were added to reactions to increase mutational frequency, while elevated ratios of dCTP and dTTP nucleotides were added in order to account for mutational bias.
  • Error-prone PCR was carried out using a low fidelity Taq polymerase and primers: Forward Primer 5'-3' : gctatcttcgctgctttgc (SEQ DI NO: 18) and Reverse Primer 5'-3' : tgtcagttcctgcaagtcttctt (SEQ ID NO: 19).
  • Final PCR products were amplified under high fidelity conditions (without Mn 2+ , equal dNTP ratios, and Phusion polymerase) and then transformed, along with digested pTMY plasmid, into EBY100 competent cells as previously described (57). Cell samples were collected post
  • Cancer cell binding assay 5xl0 5 cancer cells were resuspended in cold lxPBS with 1 mg/mL bovine serum albumin (0.1%BPBS), containing soluble inhibitor or a 1: 100 dilution soluble human matriptase antibody (Fisher Scientific). Cell solutions were incubated at 4°C for at least 3 hours (for single point binding assays) or overnight (for binding curve assays). Cells were then washed with cold 0.01% BPBS, and protein binding was measured using 1: 100 anti-mouse phycoerythrin (PE) (Invitrogen). Following incubation at 4°C for 15 min, cells were washed and analyzed using flow cytometry (BD-Accuri).
  • PE anti-mouse phycoerythrin
  • Mean cell binding (Mean-RFU) was quantified from at least 10,000 cell events and corrected for controls incubated with antibodies alone. Values were then plotted against inhibitor concentration, normalized to saturating conditions, and Kd app values were calculated by fitting graphs to a four-parameter equation (GraphPad version 6).
  • Pro-HGF activation assay A gradient of soluble inhibitors were incubated with 0.05 nM soluble matriptase in matriptase assay buffer for 30 min at room temperature.
  • Soluble Pro-HGF (125 nM) was then added to the solutions and the reactions were incubated for an additional 2 hr at room temperature. Reaction products were then boiled for 10 min at 95°C in the presence of loading dye and reducing agent, and then loaded onto a 12% SDS- polyacrylamide gel (GenScript) and subjected to electrophoresis for protein fragment separation. Protein bands were then transferred to a nitrocellulose membrane, and probed with a primary anti-HGF a-chain antibody (Abeam), followed by an anti-rabbit horse radish peroxidase (HRP) antibody (Fisher Scientific). Protein presence was then detected using SuperSignal West Femto HRP substrate (Fisher Scientific).
  • HRP horse radish peroxidase
  • MDCK cell migration/scatter assay 3xl0 3 Madin-Darby Canine Kidney (MDCK) cells were seeded in 96- well plates in 2% serum-supplemented media. Plates were cultured for 24 hr in a humidified tissue culture incubator at 37°C, 5% CO2 atmosphere. Following incubation, cells were washed twice with warm IxPBS, and 99 ⁇ serum free media was added. Reaction products from the Pro-HGF activation assay were mixed with 50 nM final concentration of respective inhibitors to quench the activation of HGF by matriptase.
  • Inhibitor was not added to the "Pro-HGF with matriptase" or "Pro-HGF alone” controls to allow uninterrupted matriptase activity. All reactions were then diluted 1 : 10 in matriptase assay buffer (R&D) and then 1 ⁇ of each reaction was added to separate wells, all in duplicate. Serum free media only served as the untreated negative control. Plates were cultured for 24 hr in a humidified incubator at 37°C, 5% CO2 atmosphere. Following incubation, cells were stained with crystal violet and then imaged at lOx magnification for qualitative assessment of cell migration in response to active HGF.
  • Cancer cell activity assay lxlO 5 cancer cells were seeded in 96-well plates in 2% serum- supplemented media. Plates were cultured for 24 hr in a humidified incubator at 37°C, 5% CO2 atmosphere. Following incubation, cells were washed twice with warm IxPBS, and serum free media was added. Soluble inhibitors were then immediately added and samples were incubated at 37°C, 5% C0 2 for 1 h. 100 ⁇ matriptase substrate (Boc-QAR-AMC; R&D) was then added to initiate the reaction and fluorescence at 380nm/460nm was measured was measured once per hour for 5 hr using a microplate reader (Synergy H4, Biotek).
  • HAI-1 is comprised of amino acids Metl to Glu449 (Genbank Accession ID: AY358969.1); KDl is comprised of amino acids Cys250 to Val303 (Genbank Accession ID: AY358969.1), and Pro-HGF is comprised of amino acids Metl to Ser728 (HGF Isoform 3, Genbank Accession ID: NP_001010932).
  • DNA encoding the open reading frame of the HAI- 1 monomer, Pro-HGF, and Fc-fusion constructs was cloned into the pCEP4 mammalian expression plasmid (Invitrogen).
  • pCEP4 vectors were amplified and transfected into adherent human embryonic kidney (HEK) cells using Lipofectamin 2000 (Fisher Scientific). Transfected HEK cells were selected using 400 ⁇ g/mL Hygromycin B (Fisher Scientific), and cultured in DMEM containing 10% FBS in a humidified incubator at 37°C, 5% CO2. Recombinant protein expression was initiated by the addition of serum-free
  • DMEM fetal calf serum
  • the KDl monomer was cloned into the pPIC9 yeast expression plasmid, transformed into the yeast strain P. pastoris, and expressed using reagents, media, and protocols exactly as previously described (52).
  • Protein-containing supernatants from HEK cells and P. pastoris was purified by Ni-NTA metal chelating chromatography (for monomer inhibitors and Pro-HGF containing a hexa-histidine-tag) or Protein A affinity chromatography (for inhibitors containing a Fc fusion), followed by size exclusion chromatography (s75 10/300 GL, s200 Increase 10/300 GL; GE Healthcare).
  • Purified protein was then characterized using SDS-polyacrylamide gel electrophoresis (SDS- PAGE) and concentrations were quantified by UV-Vis absorbance (280nm) and extinction coefficients: (HAI Fc fusion variants; 179810cm 1 M 1 , HAI monomer; STlOOcm ⁇ M 1 , KDl monomer; 11835 cm ⁇ M 1 , Pro-HGF; 149180cm 1 M 1 .
  • Purified proteins were stored in lxPBS (inhibitors) or lxPBS with 500mM NaCl (Pro-HGF) at 4°C and tested within three weeks or flash frozen with 0.01% Tween 80 for long term storage at -80°C.
  • amino acid sequence of the fusion protein used in some these experiments is shown below (SEQ NO: 4), where the sequence in bold is HAI-1, the underlined sequence in bold is the chimeric KD2/1 domain as described above, and the italicized sequence is a mouse Fc domain.
  • HAI- 1 was used as a starting scaffold for protein engineering to leverage its intrinsic ability to bind and inhibit matriptase.
  • HAI-1 is comprised of an N-terminal domain (41), an internal domain (42), a first Kunitz domain (KDl), a low-density lipoprotein (LDL) like domain, a second Kunitz domain (KD2), a transmembrane domain, and an intracellular domain (Fig. ).
  • KDl has been well established as the minimal matriptase binding domain within HAI-1.
  • KD2 has been shown to negatively regulate HAI-1 binding affinity and confer protease specificity (26, 34).
  • Yeast surface display is a well-established protein engineering technology that has been used for characterizing and screening protein-based inhibitors (40, 43-45) (Fig. 5 B) KDl and KD2 were well expressed on the surface of yeast as Aga2p mating protein fusions (Fig. 5(7). Additionally, it was shown that KDl bound to soluble matriptase with an affinity (Kd) of 13 + 2 pM, while KD2 exhibited no detectable binding (Fig. 2 C), a trend in agreement with previous results. KD2 has been suggested to retain the secondary matriptase binding site conserved from KDl (26).
  • epPCR error-prone polymerase chain reaction
  • DNA from the final sorted yeast population was isolated and sequenced to identify amino acid mutations that could confer increased matriptase binding compared to wild-type KDl and KD2 (Fig. 2 B and Fig. 12).
  • a chimeric variant that essentially was a fusion of the N-terminus of KD2 and C-terminus of KDl (clone 33, named KD2/1) was identified.
  • Select yeast-displayed variants were individually tested for binding to matriptase; only the KD2/1 chimera and wild-type KDl showed any detectable binding signal (Fig. 6).
  • a soluble, recombinant matriptase inhibitor was created.
  • the wild-type KD2 domain of full- length HAI-1 was replaced with the sequence of the engineered KD2/1 chimera.
  • the construct was fused to the crystallizable fragment (Fc) domain of an antibody, which can confers therapeutic properties including circulating half-life extension, immune system recruitment, and elevated binding affinity through avidity (49, 50).
  • This protein was termed KD1-KD2/1-FC (Fig. 3).
  • yeast- displayed KDl Fig.
  • HAI-1 design was created where the wild-type KD2 domain was replaced with a second wild-type KDl domain, termed KDlx2-Fc.
  • KDlx2-Fc wild-type KDl monomer, full-length HAI-1 monomer, and a HAI-1 Fc fusion (HAI-Fc) were also produced as controls.
  • the function of the KDl domain can be diminished by introducing an R260A point mutation, which is known to ablate matriptase binding (34).
  • this point mutation was used to create constructs containing a non-inhibitory KDl domain to allow us to parse the importance of each Kunitz domain on matriptase inhibition (Fig.
  • KD1-KD2/1-Fc is a potent and selective inhibitor of matriptase
  • the purified proteins were next evaluated for their ability to inhibit matriptase activity.
  • Each protein construct was tested using an in vitro kinetic inhibition assay. Dose response plots were generated for each inhibitor (Fig. 8 A) and inhibition constant (Ki) values were determined using equation (1) and equation (2) as previously reported (36, 38, 40). Table 1 lists the resulting Ki value for each inhibitor construct and the number of functional Kunitz domains present.
  • HAI-R260A-Fc has no detectable inhibition of matriptase due to the ablating R260A mutation that disrupts wild-type KD1 function, and also confirms that the KD2 domain or Fc domain does not participate in matriptase inhibition.
  • the KD1-R260A-KD2/1-Fc protein exhibited a Ki of 550 + 50 pM, indicating that the KD2/1 domain is a functional inhibitor of matriptase when incorporated into the HAI-1 Fc fusion protein.
  • the wild-type KD1 monomer has a Ki of 310 + 20 pM, which is in agreement to previous Ki measurements for matriptase (34, 42), and is a 30-fold more potent inhibitor relative to the full length HAI- 1 monomer. This improvement further demonstrates the negative regulation of KD2 in the context of the full length HAI-1 inhibitor.
  • Table 1 Summary of the Ki values quantified from dose response plots (Fig, 8 A) for each soluble inhibitor tested. Values were fit and calculated using equations 1 and 2 and reported as the mean and standard deviation of triplicate measurements.
  • KD1-KD2/1-Fc had the lowest Ki of 70 + 5 pM, making it the most effective inhibitor in the panel. More specifically, KD1-KD2/1-Fc demonstrates a relative
  • KD1-KD2/1-Fc The selectivity of KD1-KD2/1-Fc was tested against a panel of naturally soluble or cell anchored serine proteases, including trypsin 3 (51), urokinase (11), kallikrein 4 (30), and hepsin (28, 29); each of these proteases have a range of native affinities to wild- type HAI-L It was found that none of the proteins tested could inhibit trypsin 3 or urokinase activity (Fig. 8 B-D and Table 2).
  • KD1-KD2/1-Fc and wild-type KD1 monomer inhibit kallikrein 4 similarly, at 8.0 + 2 nM and 9.3 + 2 nM respectively; while the wild-type HAI-1 monomer more weakly inhibits kallikrein 4 with Ki values above 100 nM.
  • KD1-KD2/1-Fc and wild-type KD1 monomer inhibit hepsin with Ki values of 1.5 + 0.4 nM and 5.4 + 2 nM respectively; while the wild-type HAI-1 monomer more weakly inhibits hepsin with a Ki value of 72 + 30 nM.
  • KD1-KD2/1-Fc for matriptase remains at >1, 000-fold over trypsin 3 and urokinase, 110-fold over kallikrein 4, and 20-fold over hepsin.
  • KD1-KD2/1-Fc inhibits matriptase-mediated activation of pro-HGF
  • KD1-KD2/1-Fc The ability of KD1-KD2/1-Fc to inhibit matriptase cleavage and activation of human Pro-HGF (huPro-HGF), the natural substrate target that contributes to cancer metastasis (Fig. 1 B, 10 A-C), was tested. These results indicate that KD1-KD2/1-Fc inhibits matriptase mediated cleavage of huPro-HGF in a dose dependent manner, and qualitatively appears to be a more potent inhibitor than wild-type KDl monomer.
  • Reaction products from the huPro- HGF activation experiment were then incubated with Madin-Darby Canine Kidney (MDCK) cells to measure HGF- mediated cell migration via c-Met receptor binding and activation (11, 37, 52) (Fig. 10 D).
  • MDCK Madin-Darby Canine Kidney
  • the addition of the KD1-KD2/1-Fc treated sample reduces MDCK migration in a dose dependent manner, compared to uninhibited control samples, further supporting that KD1-KD2/1-Fc potently inhibits activation of huPro-HGF by matriptase. Additionally, KD1-KD2/1-Fc qualitatively prevents cell scattering at lower concentrations than wild- type KDl monomer.
  • KD1-KD2/1-Fc inhibits matriptase expressed on cancer cells
  • the ability of KD1-KD2/1-Fc to inhibit matriptase expressed on human cancer cell lines was tested.
  • Expression and functional activity of matriptase was confirmed on the surface of three human cancer cell lines, MDA-MB-231 (breast), A549 (lung), and PC3 (prostate), using a matriptase specific antibody and a commercial matriptase substrate.
  • Positive matriptase expression levels correlating with matriptase functional activity were identified for each cell line tested (Fig. 11 A-B).
  • KD1-KD2/1-Fc was then tested and compared with wild-type KD1 and HAI-1 monomer proteins for inhibition of fluorescent matriptase substrate activation. Dose response curves of matriptase inhibition were then generated and fit to quantify IC50 values for each inhibitor tested (Fig. 4 A). The results demonstrate that KD1-KD2/1-Fc inhibits matriptase up to 10-fold and 30-fold better compared to wild-type KD1 and HAI-1 inhibitors, respectively. KD1-KD2/1-Fc was also confirmed to bind to the surface of cancer cell lines, further confirming specific interactions with cell associated matriptase (Fig. 11 C).
  • a cancer cell invasion assay was performed to further test the ability of KD1-KD2/1-
  • Fc to inhibit cell expressed matriptase activation of huPro-HGF.
  • Invasion assays are often used to measure the phenotypic behavior of cancer cells in response to growth factor stimulation and protease inhibition involving the matriptase-HAI-1 / Pro-HGF-Met pathway (Fig. 1 B) (33, 53).
  • HEK cells were transfected to overexpress soluble huPro-HGF (Fig. 10 A-B).
  • the HEK-huPro-HGF cell line was used to construct a co-culture assay to model cancer cell invasion in response to paracrine growth factor stimulation.
  • KD1-KD2/1-Fc can potently bind to and inhibit soluble and cell expressed matriptase, and block huPro-HGF activation to impede cell migration and invasion.
  • the bivalent KD1-KD2/1-Fc fusion contains four matriptase binding domains: two wild-type KDl domains and two engineered KD2/KD1 (KD2/1) chimeric domains, accentuating its potency 120-fold relative to wild-type HAI-1 (Table 1).
  • KD2/1 chimeric domains This dramatic improvement can be attributed to replacement of the sterically regulating wild-type KD2 domain with the engineered KD2/1 matriptase -binding chimera.
  • the KD2/1 chimeric domain seemed to be important for this function, as a protein variant created by replacing the KD2 domain with another wild-type KD1 domain (KDlx2-Fc) was unable to be
  • Ki for KD1-KD2/1-Fc improves 4-fold relative to wild-type KD1 monomer, suggesting stoichiometric binding of one matriptase molecule to one functionally inhibiting domain of KD1-KD2/1-Fc.
  • This relationship is also observed in the 2-fold relative difference in Ki between wild-type HAI-1 monomer compared with HAI-Fc.
  • Stoichiometric 1 1 wild-type HAI-1 and matriptase complexes have been previously observed to occur naturally (54). This model of inhibition assumes that all four KD1-KD2/1-Fc functional domains are equally accessible for simultaneous matriptase inhibition.
  • KD1-KD2/1-FC additionally retains the highest selectivity to matriptase amongst a panel of serine proteases tested (Table 2).
  • KD1-KD2/1-Fc and wild-type KDl inhibit kallikrein 4 with low nanomolar Ki values
  • wild-type HAI-1 monomer only weakly inhibits kallikrein 4 with a Ki >100 nM.
  • This result further demonstrates the regulatory role that KD2 plays on the KDl domain within the context of full-length HAI-1. This regulation is also observed for hepsin, where KD1-KD2/1-Fc and wild-type KDl monomer inhibit hepsin more effectively compared to the wild-type HAI- 1 monomer.
  • KD1-KD2/1-Fc protease selectivity profile Summary of the Ki values quantified from dose response plots (Fig. 8 B) for each soluble protease and inhibitor tested.
  • KD1 WT wild- type KD1 monomer
  • HAI-1 WT wild- type HAI-1 monomer. Values were fit and calculated using equations 1 and 2 and reported as the mean and standard deviation of triplicate measurements. Appoximate fold selectivity values for wild-type KD1 monomer and HAI-1 monomer are reported relative to KD1-KD2/1-Fc.
  • KD1-KD2/1-Fc inhibits matriptase activation of the pro-domain form of HGF, with reduced MDCK cell scattering at lower inhibitor concentrations than wild-type KD1 monomer (Fig. 10).
  • KD1-KD2/1-Fc efficacy is further observed by a 10-fold greater inhibition of matriptase activity on cancer cells relative to wild-type KD1 monomer (Fig. 4 A).
  • KD1-KD2/1-Fc also significantly reduces lung and breast cancer cell invasion in vitro (Fig. 4 B).
  • the extent of reduced invasion aligns with previous inhibition results using this standard invasion model (33, 53).
  • the invasion assay also included media containing 2% fetal bovine serum to maintain HEK cell viability, identified to contain significant levels of active proteases capable of cleaving commercial matriptase substrate.
  • the KD1-KD2/1-Fc construct has a Ki for matriptase of 70 + 5 pM, which is amongst the tightest Ki values measured for protein based matriptase inhibitors.
  • Ki 310 pM + 20 pM
  • KD1-KD2/1-FC protein fulfills several attractive design criteria.
  • use of the native HAI- 1 as a starting point for therapeutic development leverages the affinity and specificity of the natural inhibitor.
  • replacing the KD2 domain with an active matriptase-binding domain is expected to be minimally perturbing to native HAI-1.
  • fusion of the engineered construct to an Fc domain creates a bivalent protein, in this case, resulting in 4 matriptase binding sites that improves protease inhibition.
  • fusion to an Fc domain is expected to increase serum half-life through increased molecular weight and FcRn-mediated recycling (50) requiring less frequent therapeutic dosing, and allowing manufacturing processes that are similar to antibodies.
  • Matriptase imaging experiments have also suggested that cell anchored HAI-1 can serve as a natural reservoir for secreted proteases, effectively increasing their local concentration and activity at the leading edge of cancer invasion (55, 56). High affinity inhibitor binding is therefore critical to effectively outcompete the interaction of proteases to native cell surface HAI-1.
  • the engineered matriptase binding protein described here thus has the potential to function as a HAI- 1 "decoy" in cancer and other disorders where matriptase underlies disease pathophysiology.
  • adenocarcinoma Overexpression of matriptase with concomitant loss of its inhibitor, hepatocyte growth factor activator inhibitor- 1. Cancer Epidemiol Biomarkers Prev
  • HAI-1 hepatocyte growth factor activator inhibitor- 1
  • HAI-1 hepatocyte growth factor activator inhibitor type 1
  • Zhao B, et al. (2013) Crystal structures of matriptase in complex with its inhibitor hepatocyte growth factor activator inhibitor-1. J Biol Chem 288: 11155-11164.
  • Cirino PC Mayer KM, Umeno D (2003) Generating mutant libraries using error- prone PCR. Methods Mol Biol 231(12):3-9.
  • HAI-1 hepatocyte growth factor activator inhibitor type 1
  • YPD media 20 g/L glucose, 20 g/L peptone, and 10 g/L yeast extract
  • SD-CAA media 20 g/L dextrose, 6.7 g/L yeast nitrogen base lacking amino acids, 5.4 g/L Na 2 HP0 4 , 8.6 g/L NaH 2 P0 4 H20, and 5 g/L Bacto casamino acids
  • SG-CAA media same as SD-CAA, but 20 g/L galactose is substituted for dextrose.
  • BMMY, BMGY media, and RDB plates for P. pastoris strain GS115 were prepared exactly as described. 37
  • Complete growth media Dulbecco's modified Eagle medium (Fisher Scientific), 10% fetal bovine serum (FBS) (Fisher Scientific).
  • Cell lines used include: Human Embryonic Kidney (HEK) cells, PC3 (prostate cancer), MDA-MB-231 (breast cancer), and A549 (lung cancer) - (ATCC).
  • Biosensor Molecular Cloning The DNA sequence for each linker design was based upon the natural matriptase cleavage sequence found within the human pro-macrophage stimulating protein (Asp473 to Trp494; Genbank Accession ID: 4485). DNA encoding for each linker was genetically fused to the "A" domain using PCR, and incorporated into the ddRFP construct with EcoRI and Hindlll restriction enzyme sites into the pB AD vector 26 . A C-terminal hexahistidine tag was included in each construct for downstream purification and western blot detection.
  • Biosensor Production Following transformation into DH10B electrocompetent E.coli cells, each matriptase biosensor construct was expressed and purified. Briefly, transformed cells were expanded in Luria broth with 0.1 mg/mL ampicillin at 37°C, and biosensor protein expression was induced by addition of 0.2% (v/v) arabinose. Following overnight culture, each protein was extracted from E. coli inclusion bodies using Bacterial Protein Extraction Reagent (B PER- Thermo Fisher) and purified via Ni-NTA metal chelating chromatography. Absorbance values at 570nm were measured to quantify concentration using a previously reported extinction coefficient (48,300M 1 cm 1 ). 10
  • Matriptase cleavage screen 1 ⁇ of each biosensor was added to 10 nM, 1 nM, or 0.1 nM recombinant human matriptase (R&D Systems) using manufacturer's buffers in a 384-well plate at room temperature. Each biosensor without matriptase (buffer only) served as a negative background control. Fluorescent output (535nm/605nm) was monitored over time and initial velocity values were quantified by first correcting for conditions without matriptase and then converting the initial reaction rate (RFU/hr) to matriptase velocity (nM/s) using a determined standard curve.
  • R&D Systems 0.1 nM recombinant human matriptase
  • Protease selectivity screen 1 ⁇ of B4, B8, or B9 were separately added to 10 nM recombinant human trypsin 3, urokinase, kallikrein 4, and hepsin (all from R&D Systems) using manufacturer's buffers in a 384-well plate at room temperature. Each biosensor without protease (buffer only) served as a negative background control. Fluorescent output measurement and initial velocity values were quantified for each condition as described above.
  • Michaelis-Menten assay Varying concentrations of B 4 (0 to 50 ⁇ ) was added to 10 nM recombinant human matriptase using manufacturer's buffers in a 384-well plate at room temperature. B4 without matriptase (buffer only) served as a negative background control. Fluorescent output measurement and initial velocity values were quantified for each condition as described above. Final velocity values were plotted and fit to a Michaelis- Menten curve using Prism GraphPad software to quantify kinetic parameters, including: K m , Vmax, and k cat-
  • proteins were transferred to a nitrocellulose membrane, washed once with Milli-Q water and blocked with 5% bovine serum albumin (BSA) in IxTBST (lmL Tween 20: 1L Tris-buffer saline) buffer for one hour at room temperature. Membranes were then transferred to IxTBST containing 5% dry milk (BioRad) and (1 :10,000) dilution primary anti-His tag antibody (GenScript) for one hour at room temperature.
  • BSA bovine serum albumin
  • PAGE electrophoresis
  • concentration was measured by UV-Vis absorbance (280 nm) and extinction coefficient 11835 cm ⁇ M 1 .
  • Purified protein was stored in lxPBS at 4°C (short term storage) or flash frozen with 0.01% Tween 80 at -80°C (long term storage).
  • the human KD1-R260A variant was created using standard overlap PCR assembly site-directed mutagenesis techniques. Human KD1 gene products were cloned into the pTMY yeast display vector as previously described 37 using Nhel and Mlul restriction sites. Plasmids were transformed into EBY100 yeast cells using electroporation, expanded in SD-CAA media at 30°C, and then induced for surface protein display using SG-CAA media at 20°C.
  • Antibodies to measure yeast surface expression (1 :250 dilution of anti-HA, mouse primary antibody (Fisher Scientific) and 1: 100 dilution of anti-HAI-1, rabbit primary antibody (Fisher Scientific)) were added and incubated with yeast for 30 min at room temperature followed by washing cells with manufacturers matriptase assay buffers (R&D Systems). Primary antibody binding was then detected by incubating yeast with a 1: 100 dilution of anti-mouse phycoerythrin-PE
  • Matriptase Inhibition Assay 10 nM matriptase was added to purified KD1 soluble inhibitor (final concentrations ranging 0 - 750 nM) in matriptase assay buffer. Immediately, 1 ⁇ purified B4 was added to initiate the reaction. Yeast surface display matriptase inhibition was carried out using the same matriptase and substrate conditions as above, except induced yeast displaying inhibitor domains were first counted (10 - 10 8 yeast cells/sample) and incubated with matriptase prior to addition of B4. Kinetic monitoring of fluorescent output from B4 was performed using methods and conditions described above.
  • Cancer Cell Matriptase Expression 5 x 10 5 cancer cells were resuspended in cold
  • Cell solutions were incubated at 4°C for 2 h, washed with cold 0.01% BPBS, and stained antibody binding using a 1 : 100 dilution of anti-mouse phycoerythrin (PE) (Invitrogen). Following 15 min incubation at 4°C, cells were washed and analyzed for binding using flow cytometry (BD CSampler software). Mean cell binding (Mean-RFU) was quantified from at least 10,000 cell events and subtracted from fluorescent background labeling with secondary antibodies alone.
  • PE anti-mouse phycoerythrin
  • Cancer Cell Matriptase Activity Assay 1 x 10 5 cancer cells were seeded in 96-well plates in 2% fetal bovine serum- supplemented media. Plates were cultured for 24 h in a humidified tissue culture incubator at 37 °C, 5% CO2 atmosphere. Following incubation, cells were washed twice with warm IxPBS to remove residual serum, and serum free media (DMEM alone) was added. ⁇ purified B4 was immediately added to each condition and incubated at 37°C, 5% CO2 for 1 h. B4 in serum free media alone served as a control condition. Plates were maintained at 37°C, 5% CO2 and fluorescent output (535nm/605nm) was monitored every hour for at least 5 h.
  • Rate of fluorescence change over time was quantified by fitting a linear curve using GraphPad software and averaging duplicate samples. Separately, the same experiment was performed using commercial matriptase substrate, Boc-QAR-AMC (R&D), and fluorescent output (380nm/460nm) was measured over time.
  • Pro-MSP pro- macrophage stimulating protein
  • the matriptase recognition site of pro-MSP has been well established and includes the sequence Ser-Lys- Leu-Arg-Val-Val-Gly-Gly (P4-P4' , N to C termini), with Arg483-Val484 representing the scissile bond.
  • P4-P4' the sequence Ser-Lys- Leu-Arg-Val-Val-Gly-Gly
  • Arg483-Val484 representing the scissile bond.
  • each biosensor construct was then characterized for its ability to detect matriptase activity.
  • Fig. 17 A demonstrates a typical time course plot for each construct, in which fluorescent emission is measured over time in the presence or absence of matriptase.
  • fluorescence decreases over time in the presence of matriptase, but is retained over time in the absence of matriptase.
  • constructs B4 through B9 portray these ideal fluorescent properties
  • B3 exhibits no change in fluorescence over time with matriptase, while BIO reveals low fluorescence.
  • Target selectivity is an important consideration in protease biosensor design for measuring protease activity in a heterogeneous environment.
  • a time course experiment was performed with a panel of serine proteases including trypsin 3 29 , urokinase, 30 kallikrein 4, 31 and hepsin, 32 in addition to matriptase.
  • the resulting initial velocity values for each biosensor and protease tested were compared to matriptase activity (Fig. 17 D).
  • B4 has a linear activity response to matriptase concentration and follows ideal Michaelis-Menten kinetics, with a quantified K m of 8.17 x lO 6 M and a k ca t/K m of 2.09 x 10 7 M _1 s _1 . These parameters indicate high specificity and highly efficient turnover by matriptase. B4 was next tested for its ability to detect activity of matriptase expressed on tumor cells and inhibition in the context of a natural protein. The expression profile of endogenous matriptase on three human cancer cell lines, PC3 (prostate cancer), A549 (lung cancer), and MDA-MB-231 (breast cancer) was characterized.
  • HAI-1 matriptase hepatocyte growth factor activator inhibitor type-1
  • KDl was shown to inhibit matriptase cleavage of the B4 biosensor with a dose dependence on inhibitor concentration (Fig. 18 B). As expected, complete inhibition matched conditions without matriptase, while no inhibition approached conditions without inhibitor, with a 5 -fold dynamic range for the assay.
  • B4 biosensor could detect matriptase inhibition by a yeast surface displayed KDl inhibitor.
  • Yeast surface display is an established protein engineering technology for characterizing and screening protein-based protease inhibitors. 35 , 36 .
  • most protease screening methods rely on inhibitor-protease binding, rather than functional inhibition as a criteria for selection.
  • B4 can be used to detect matriptase inhibition by yeast displayed proteins
  • a future application could utilize B4 as a functional screening tool for identifying improved protease inhibitor candidates from a library of yeast displayed variants.
  • KDl was displayed on the surface of S. cerevisiae yeast cells as a fusion to the agglutinin mating protein Aga2p.
  • KD1-R260A positon 260
  • This negative control protein will account for non-specific interactions with the native yeast surface proteins.
  • KD1 and KD1-R260A were both expressed on the surface of yeast, but only KD1 binds to matriptase, as expected. It was confirmed that yeast- displayed KD1, but not KD1-R260A, inhibits activation of the commercial matriptase substrate (Boc-QAR-AMC). Finally, using the B4 biosensor (Fig. 18 C), distinct matriptase inhibition by yeast-displayed KD1 with a 5 -fold dynamic range compared to KD1-R260A was observed.
  • a protease biosensor created from dimerization-dependent fluorescent protein domains provides beneficial properties compared to small molecule -based strategies including easy and low cost production, desirable spectral properties compatible with standard microscopes and flow cytometers, and robust activity in a range of assay conditions.
  • the ddRFP system is modular and can potentially be adapted for measuring activity of alternative protease targets.
  • Our efforts showed that the linker that joins the fluorescent protein domains was critical to its success as a biosensor component: a linker that is too short is not efficiently cleaved, and one that is too long exhibits nonspecific or promiscuous cleavage, or weak fluorescence.
  • Biosensor 4 containing the linker sequence RSKLRVGGH, exhibited the highest matriptase selectivity, linker specific cleavage, stability, and follows ideal Michaelis-Menten kinetic behavior.
  • Application of the B4 sensor enabled measurement of matriptase inhibition by both soluble and yeast-displayed inhibitor proteins, with up to a 5-fold dynamic range. Additionally, the B4 sensor was able to detect matriptase activity expressed by human cancer cell lines. Due to its ability to measure matriptase inhibition by soluble or cell surface proteins, the B4 biosensor can serve as a drug screening tool to identify matriptase inhibitors, as well as characterizing lead inhibitor candidates. In addition, detection of soluble matriptase and matriptase expressed on cancer cells also provides the opportunity to utilize B4 for diagnostic purposes.
  • Kallikrein 4 is a proliferative factor that is overexpressed in prostate cancer. Cancer Res. 67, 5221-5230.
  • Yeast media/plates (YPD media) 20 g/L glucose, 20 g/L peptone, and 10 g/L yeast extract; (SD-CAA media) 20 g/L dextrose, 6.7 g/L yeast nitrogen base lacking amino acids, 5.4 g/L Na2HP04, 8.6 g/L NaH2P04 ⁇ H20, and 5 g/L Bacto casamino acids; (SG-CAA media) same as SD-CAA, but dextrose is substituted for 20 g/L galactose; (Low pH, SD-CAA media) same as to SD-CAA, but phosphates reagents were substituted for 13.7 g/L sodium citrate dihydrate, 8.4 g/L citric acid anhydrous, and adjusted to pH 4.5.
  • YPD media 20 g/L glucose, 20 g/L peptone, and 10 g/L yeast extract
  • SD-CAA media 20 g/L dextrose, 6.7
  • BMMY, BMGY media, and RDB plates for P. pastoris strain GS115 were prepared exactly as described [42].
  • Mammalian media/cells (10% complete growth media) 10% fetal bovine serum (FBS) (Fisher Scientific)-containing Dulbecco's modified Eagle medium (Fisher Scientific); Cell lines used include: Human Embryonic Kidney (HEK) cells.
  • FBS fetal bovine serum
  • HEK Human Embryonic Kidney
  • KD1 variant cloning and library construction KDl consists of amino acids (Cys250 to Val303; Genbank Accession ID: AY358969.1) and was inserted into the pTMY yeast display vector previously described [42] using Nhel and Mlul restriction sites. Final plasmids were transformed into EBY100 yeast cells using electroporation, expanded in SD- CAA media at 30°C, and then induced for surface protein using SG-CAA media at 20°C. Note, all other yeast displayed proteins were cloned and prepared in this way, see below for gene details. Proper expression (1:250 dilution of anti-HA, mouse primary antibody (Fisher Scientific)) and fold (1: 100 dilution of anti-HAI-1, rabbit primary antibody (Fisher
  • KDl Library Round 1 was constructed using error-prone PCR (epPCR) as previously reported [43].
  • epPCR error-prone PCR
  • variable concentration of Mn 2+ 0.075mM and 0.15mM
  • epPCR was carried out using a low fidelity Taq polymerase and primers: Forward Primer 5 '-3' : gctatcttcgctgctttgc (SEQ ID NO: 18) and Reverse Primer 5'-3' : tgtcagttcctgcaagtcttcttt (SEQ ID NO: 19).
  • Yeast surface display library screening conditions Transfromed EBY100 yeast cells were counted, washed, and then mixed with soluble matriptase (final concentration 0 - ⁇ ) and matriptase assay buffer, at volumes accounting for ligand depletion. Reactions were incubated until reaching equilibrium, up to 48 hours at room temperature (RT).
  • Samples were then washed and stained for yeast expression (1: 100 dilution of anti-mouse phycoerythrin PE (Invitrogen)) and matriptase binding (1 : 100 dilution of anti-His fluorescein isothiocyanate FITC (Bethyl)). Samples were incubated a 4°C for 15min, washed, and maintained on ice until loading onto a flow cytometer for analysis (BD Accuri) or a fluorescent activated cell sorter (FACS) for library sorting (BD Aria II). Sorted cells were recovered in SD-CAA liquid media and incubated at 30°C overnight or until saturated (O.D. 4-8), and yeast culture protein expression was then induced (O.D.
  • E. coli cells electrocompetent E. coli cells for plasmid amplification, and then lysed for DNA extraction (Miniprep; Fisher Scientific) and sequencing.
  • HAI-1 consists of amino acids (Metl to Glu449;
  • Genbank Accession ID: AY358969.1 and KDl consists of amino acids (Cys250 to Val303; Genbank Accession ID: AY358969.1).
  • DNA encoding the open reading frame of each gene was cloned into the pCEP4 mammalian secreting expression plasmid (HAI- 1 monomer and Fc fusion constructs) or pPIC9 yeast secreting expression plasmid (KDl monomer), amplified using DH10B electrocompetent E. coli cells, and confirmed by sequencing.
  • pPIC9 cloning, transformation into P. Pastoris, and protein expression was completed using reagents, media, and protocols exactly as previously described [42].
  • Inhibitor genes were cloned into the pCEP4 vector using the Notl and Hindlll restriction sites, and a c-terminal 6- histidine tag (monomer inhibitors) or mouse IgG2a Fc domains was genetically linked using the Notl and Xhol restriction sites.
  • pCEP4 vectors were amplified and transfected into adherent human embryonic kidney (HEK) cells using Lipofectamin 2000 (Fisher Scientific).
  • Transfected HEK cells were selected for using 400 ⁇ g/mL Hygromycin B (Fisher Scientific), and expanded in 10% fetal bovine serum (FBS)-containing Dulbecco's modified Eagle medium (DMEM), and incubation in a humidified tissue culture incubator at 37°C, 5% C02 atmosphere. Soluble protein expression was initiated by the addition of serum free DMEM performed for one week. Protein expressed from HEK cells and P. Pastoris was separately collected and purified on Ni-NTA (his-tag monomer inhibitors) or Protein A (Fc fusion inhibitors) chromatography, followed by size exclusion chromatography.
  • Ni-NTA his-tag monomer inhibitors
  • Protein A Protein A
  • Purified protein was then characterized using polyacrylamide gel electrophoresis (PAGE) and concentration was measured by UV-Vis absorbance (280nm). Purified proteins were stored in lxPBS (inhibitors) at 4°C or flash frozen with 0.01% Tween 80 for long term storage at -80°C.
  • PAGE polyacrylamide gel electrophoresis
  • DH10B electrocompetent Ecoli cells a dimerization dependent-matriptase sensitive red fluorescent protein biosensor construct design was expressed and purified. Briefly, transformed cells were expanded in LB with O.lmg/mL ampicillin culture at 37°C and biosensor protein expression was induced by addition of 0.2% (v/v) arabinose and cultured overnight. Following the expression period, each matriptase biosensor was successfully extracted from E.
  • Protease inhibition assay ⁇ matriptase (R&D) was first added to soluble inhibitor (final concentrations ranging 0-500nM) containing matriptase assay buffer. ⁇ soluble matriptase substrate (Biosensor 4) was added to initiate the reaction. Fluorescent output was measured over time at 535nm/605nm using a kinetic microplate reader to determine protease activity, RFU/s. Relative velocities were plotted against inhibitor concentration, and inhibition values, IC50, were determined by fitting each curve to the competitive inhibition binding equation (1) Results/Discussion for Example 3
  • the first engineering strategy was to enhance the matriptase affinity of the wild-type KD1 domain.
  • the crystal structure analysis of wild-type KD1 monomer in complex with the matriptase catalytic domain has revealed that the primary binding interface of KD1 at amino acid position 258 to 261 is comprised of the amino acid sequence "Arg-Cys-Arg-Gly" (SEQ ID NO: 16) [30], [31].
  • the two critical arginine side chains are found to interact specifically within the matriptase active subsite pocket residues, effectively inhibiting matriptase activity [30].
  • the glycine residue within the binding motif is believed to allow flexibility and close proximity of wild-type KD1 to bind tightly to matriptase.
  • a secondary binding interface (Arg265, Asn286, and Asn289) is believed to exist in the C-terminal region of wild- type KD1, which further improves the matriptase inhibition constant to reach approximately 300pM [28], [31]. It is hypothesized that inserting random mutations placed throughout the wild-type KD1 gene sequence could introduce novel residues that further participate in matriptase binding and inhibition and could therefore enhance the inhibition constant to low pM levels; effectively outcompeting wild- type HAI-1 -matriptase binding and Pro-HGF-matriptase activation.
  • yeast surface display is an extremely robust and well-established protein engineering technology that has been used for characterizing and screening protein-based inhibitors [34]-[38] (FIG. 28, A).
  • epPCR error prone polymerase chain reaction
  • the initial mutation frequency averaged 1-4 amino acid mutations per gene with over 50% of the library folded and expressed (FIG. 20, A).
  • the KD1 Library; Round 1 was then sorted using directed evolution by yeast display and fluorescent activated cell sorting (FACS) (FIG. 29). Sorting conditions were comprised of a combination of direct equilibrium binding to matriptase (Sort 1 to 3) as well as off rate conditions with soluble wild-type HAI-1 monomer competitive inhibitor (Sort 4-6).
  • Equilibrium sorts involved incubation with decreasing concentration of soluble matriptase for Sort 1 (500pM), Sort 2 (250pM), and Sort 3 (lOOpM).
  • Competitive off rate sorts involved first incubating cells with lOnM matriptase, then washing away non-binding matriptase prior to incubation with increasing concentration of soluble wild-type HAI- 1 monomer for increasing durations for Sort 4 ( ⁇ HAI-1, 15hr), Sort 5 (300nM HAI-1, 20hr), and Sort 6 (300nM HAI-1, 42hr).
  • yeast were analyzed using FACS for yeast displayed KDl variants that bound to matriptase and were then collected and cultured for repeating sorting rounds of directed evolution (FIG. 29).
  • the KD1 Library; Round 2 was then sorted using directed evolution by yeast display and FACS, as previously mentioned (FIG. 29). Sorting conditions were comprised of a combination of direct equilibrium binding to matriptase (Sort 1) as well as competitive binding with an engineered soluble matriptase substrate, termed "B4" (Sort 2-5). Equilibrium sorts involved incubation with ⁇ matriptase and isolating cells that retained binding and expression of KD1 variants.
  • N254K (FIG. 23, C) reveals that S277R may help alleviate steric clashes that arise from the bulky side chain of N254Y with adjacent KD1 residues to possibly promote pie stacking interactions within KD1; further stabilizing the orientation of the primary binding motif region.
  • Yeast displayed KD1 mutants demonstrate minor improvement in direct binding to matriptase compared with wild-type KD1:
  • KD1 clones comprised of: N254Y, N254K, S277R, N254Y/S277R, N254K/S277R, and
  • FIG. 24, B a greater difference in matriptase binding with competitive substrate was observed for some KD1 variants.
  • yeast displayed KD1 variants with ⁇ soluble matriptase
  • increasing concentration of soluble matriptase substrate B4 matriptase binding using FACS was measured and the substrate concentration required to decrease binding 50% (IC50) was quantified; with a higher IC50 indicating a tighter matriptase binding interaction of the KD1 variant.
  • IC50 substrate concentration required to decrease binding 50%
  • KD1 variants have greater binding values due to higher overall expression levels on yeast.
  • the matriptase binding to yeast expression, for varying concentrations of B4 competing matriptase substrate was normalized (FIG. 24, D).
  • the next step involved designing and developing soluble constructs derived from the HAI-1 inhibitor protein that include the engineered KDl domain mutations to improve matriptase binding affinity and circulating half-life. This approach would also test if functional improvements could be observed or altered for the KDl variants when expressed in a soluble inhibitor context.
  • the first goal was to extend the circulating half-life of wild- type KDl, since literature has previously reported that the 6kDa wild-type KDl domain monomer has a very poor circulating half-life of only 20 minutes, greatly hindering its therapeutic efficacy [32]. It was proposed that this short coming could be remedied by fusing wild- type KDl to the crystallizable fragment (Fc) of a mouse IgG2a antibody.
  • Fc fusions have been demonstrated to improve protein properties including: circulating half- life extension, immune system recruitment, and elevated binding affinity through avidity [39], [40], [41]. Due to concern of direct fusion of the large, 50kDa Fc domain and imposing steric hindrance on the KDl domain function a panel of KDl Fc fusion constructs with varying Gly4Ser linker lengths (lx, 5x and l lx) were designed to alleviate any steric constraints (FIG. 25, A).
  • each mutation type was incorporated into the KDl monomer, HAI-1 "full length" monomer, or full length Fc fusion formats (FIG. 26, A). This approach would allow us to test the context dependence of the engineered mutations on matriptase inhibition affinity.
  • Each construct was expressed from a eukaryotic expression system and purified using affinity and size exclusion chromatography. All mutant construct designs were successfully purified at sufficient levels for testing (FIG. 26,B ).
  • Soluble KDl-Fc fusion constructs demonstrate weaker inhibition of matriptase compared with wild-type KDl:
  • the next step involved testing the ability of each soluble inhibitor construct design to inhibit soluble matriptase from Biosensor 4 substrate activation.
  • the resulting IC50 values were then graphed for each inhibitor for each design tested for comparison to wild-type.
  • the results of the KDl Fc fusion matriptase inhibition profile demonstrates that as the Gly4Ser linker length increases, the matriptase inhibition IC50 increases (FIG. 27, A). This result suggests that the extended linker is compromising the KD1 domain fold or orientation, and thus preventing its ability to potently inhibit matriptase activity.
  • wild-type KD1 monomer demonstrates the greatest potency overall.
  • the reason for this elevated potency is mentioned in literature and is likely due to the lack of the sterically hindering wild-type KD2 domain that exists within the HAI- 1 "full length" protein (FIG. 19, B).
  • KD1 has been well established as the minimal required binding domain within HAI-1
  • KD2 has been shown to negatively regulate HAI-1 binding affinity and confer protease specificity [29], [30]. Therefore, because wild-type KD1 monomer is free of the regulating KD2 domain, it can more potently bind and inhibit matriptase activity, driving down its IC50 value.
  • HAI-1 hepatocyte growth factor activator inhibitor- 1
  • HAI-2 Hepatocyte growth factor activator inhibitor-2
  • HAI-2 Hepatocyte growth factor activator inhibitor type 2
  • HAI-1 hepatocyte growth factor activator inhibitor type 1
  • Hepatocyte growth factor activator inhibitor type 1 is a specific cell surface binding protein of hepatocyte growth factor activator (HGFA) and regulates HGFA activity in the pericellular microenvironment.
  • HGFA hepatocyte growth factor activator
  • Hepatocyte growth factor activator inhibitor type 1 suppresses metastatic pulmonary colonization of pancreatic carcinoma cells

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Abstract

L'invention concerne, entre autres choses, un inhibiteur à haute affinité de la protéase de matriptase. Dans certains modes de réalisation, le polypeptide comprend un premier domaine de Kunitz qui est identique à au moins 90 % de toute la longueur contiguë de la fusion KD2/1 de la SEQ ID NO : 1. L'invention concerne également des procédés d'inhibition d'une protéase de matriptase et des procédés de traitement d'une maladie ou d'une affection liée à la matriptase chez un sujet.
PCT/US2018/045431 2017-08-08 2018-08-06 Inhibiteur de matriptase modifié par haute affinité Ceased WO2019032472A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPWO2020246567A1 (fr) * 2019-06-05 2020-12-10
WO2022133069A1 (fr) * 2020-12-17 2022-06-23 Disc Medicine, Inc. Inhibiteurs peptidomimétiques de la matriptase 2 et leurs utilisations

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003066824A2 (fr) * 2002-02-07 2003-08-14 Aventis Behring Gmbh Peptides du domaine kunitz fusionnes a l'albumine
US20040171794A1 (en) * 2003-02-07 2004-09-02 Ladner Robert Charles Kunitz domain peptides
WO2005021557A2 (fr) * 2003-08-29 2005-03-10 Dyax Corp. Inhibiteurs de protease poly-pegylee
US20080274969A1 (en) * 2002-02-07 2008-11-06 Novozymes Delta Limited Albumin-Fused Kunitz Domain Peptides

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003066824A2 (fr) * 2002-02-07 2003-08-14 Aventis Behring Gmbh Peptides du domaine kunitz fusionnes a l'albumine
US20080274969A1 (en) * 2002-02-07 2008-11-06 Novozymes Delta Limited Albumin-Fused Kunitz Domain Peptides
US20040171794A1 (en) * 2003-02-07 2004-09-02 Ladner Robert Charles Kunitz domain peptides
WO2005021557A2 (fr) * 2003-08-29 2005-03-10 Dyax Corp. Inhibiteurs de protease poly-pegylee

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPWO2020246567A1 (fr) * 2019-06-05 2020-12-10
EP3981428A4 (fr) * 2019-06-05 2023-07-12 Chugai Seiyaku Kabushiki Kaisha Substrat de protéase et polypeptide comprenant une séquence de clivage de protéase
JP7716979B2 (ja) 2019-06-05 2025-08-01 中外製薬株式会社 プロテアーゼ基質、及びプロテアーゼ切断配列を含むポリペプチド
WO2022133069A1 (fr) * 2020-12-17 2022-06-23 Disc Medicine, Inc. Inhibiteurs peptidomimétiques de la matriptase 2 et leurs utilisations

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