HIGH AFFINITY ENGINEERED MATRIPTASE INHIBITOR

CROSS-REFERENCING

This application claims the benefit of US provisional application serials nos.

62/542,733, filed Aug 8, 2017, and 62/549,376, filed Aug 23, 2017, which applications are incorporated herein for all purposes.

GOVERNMENT RIGHTS

This invention was made with Government support under contract CA151706 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

High expression and dysregulated activity of the type II, membrane-anchored serine protease matriptase in the local tumor environment has been shown to correlate with poor patient prognosis in many human cancers including breast, colorectal, pancreatic, cervical, and prostate cancers (1-10). This dysregulation is partly driven by the high proteolytic processing and turnover of pro-hepatocyte growth factor (Pro-HGF) (9, 11) to a form of HGF that activates its cognate receptor, c-Met (12) (Fig. 1 A). 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.

Furthermore, 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).

Although the correlation of matriptase overexpression, dysregulation, and disease progression is well-established, effective matriptase inhibitors are lacking, highlighting an important clinical need.

Matriptase naturally functions in developmental pathways as well as in tissue regeneration (19-23). The activity of matriptase is regulated in healthy tissue by the serine protease inhibitor hepatocyte growth factor activator inhibitor type-1 (HAI-1) (Fig. 1 A). HAI- 1 is primarily expressed on the surface of epithelial cells and naturally blocks the substrate-activating properties of matriptase (24-26), as well as other structurally related proteases such as the hepatocyte growth factor activator (HGFA) (9, 27), hepsin (28, 29), and 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). As such, 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).

Inhibition of matriptase-driven cancer progression has been proposed as an attractive strategy for cancer therapy. In one study, induced cell surface expression of HAI- 1 within the tumor environment of an orthotopic adenopancreatic cancer model resulted in reduced tumor size and eliminated metastatic nodule formation (2). In another study, the addition of soluble HAI-1 was shown to significantly lower pro-HGF activation and reduce breast cancer cell invasion in vitro (33), highlighting recombinant HAI-1 as a therapeutic approach. However, the therapeutic utility of HAI- 1 is ultimately limited by its nanomolar inhibition constant to matriptase. In contrast, the protease inhibitor first Kunitz (KD1) sub-domain of HAI-1 (Fig. 1 A) (26, 34) has been shown to inhibit matriptase activity with significantly greater potency than full-length HAI-1 (35). However, the small molecular weight of the

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.

Alternative approaches to develop matriptase inhibitors include synthetic small molecules (36, 37), peptides (38), monoclonal antibodies (39), and constrained peptide scaffolds (40). While each strategy generated molecules that bound to and inhibited matriptase activity, none address all of the reported therapeutic limitations. SUMMARY

Provided herein, among other things, is a high affinity inhibitor of the matriptase protease. In some embodiments, 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.

In some embodiments, 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. 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. When dimerized, 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. In some embodiments, 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.

Also provided is a biosensor. In some embodiments 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. In these embodiments, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

Fig. 1. (A) 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. (C) Matriptase equilibrium binding of yeast-displayed KDl wild- type (K _d = 13 + 2 pM), KD2/1 chimera (K _d = 220 + 30 pM), and KD2 wild-type (no binding); reported as mean and standard deviation values. (D) Yeast-displayed KD domains that bind matriptase also inhibit its activity. Bar graphs indicate quantified matriptase activity for varying number of yeast cells. Scheme is the same as in panel C. Significance quantified with pair wise Γ-test; *p<0.0001, **p<0.0003, ***p<0.0004, ****p<0.0024. From top to bottom, SEQ ID NOs: 1-3.

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 Γ-test;

*p<0.008, **p<0.05, ***p<0.017, ****p<0.054. Fig. 5: (A) 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. Segments of the binding motif from KDl were introduced into the same region as KD2, generating "KD2-graft 1" and "KD2-graft 2". (B) Schematic depiction of yeast surface display, a powerful protein engineering tool used to measure protease-inhibitor interactions. Fluorescein isothiocyanate (FITC), Phycoerythrin (PE). (C) Yeast-displayed domains characterized for expression and polyclonal anti-HAI-1 antibody binding on the yeast surface. Each scatter plot represents 10,000 live yeast interrogated for their ability to bind antibodies that recognize yeast expression tags

(Hemagglutinin- HA or cmyc) or HAI-1 specific epitopes. 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.

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: (A) Schematic of huPro-HGF which includes a five domain a-chain (N- terminal domain and four Kringle (K) domains), the β-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 β chains. (B) The histidine tag enables detection in a western blot to confirm molecular weight of approximately 80 kDa (non-reduced), also verified by FPLC. (C) Inhibition of matriptase activation of huPro-HGF was tested with soluble 50 pM matriptase incubated with varying doses (0, 12.5, 25, 50, 500, 5000 pM) of KD1-KD2/1- Fc or KD1 wild-type monomer (WT-KD1) prior to addition of purified 125 nM huPro-HGF. Reaction products were separated with SDS-PAGE under reducing conditions and western blot stained with an anti-HGF a chain antibody to detect the presence of Pro-HGF (inactive species) and HGF a-chain (active species). Note: β-chain is not detected due to staining conditions. 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.

Quantified matriptase activity levels, with (striped) or without (solid) KD1-KD2/1-Fc inhibitor, compared to media only conditions (brown). * p<0.02 vs cell only condition (C) Equilibrium binding assay of soluble KD1-KD2/1-Fc binding to matriptase expressed on PC3 and MDA-MB-231 cancer cell lines. Binding fluorescence was measured using flow cytometry for PC3 (Kd _app = 10 + 2 nM) and MDA-MB-231 (Kd _app = 7 + 2 nM). Data was fit using GraphPad Prism software to quantify apparent affinities (Kd _app ) which are reported as mean and standard deviation.

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; Clone 33 (KD2/1): SEQ ID NO: 2.

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; KD2/1 (structure): SEQ ID NO: 2; KDl mutation sites: SEQ ID NO: 3. Fig. 14 is a table showing the summary of the sequences 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 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. N-term = N-terminus; Kl-4 = Kringle domains 1-4; SPH = Serine Protease Homolog. Sequences from top to bottom: SEQ ID NOs.: 8-15. 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.). (C) Western blot bands correspond to three possible reaction products detected through the C-terminal hexahistidine-tag: uncleaved biosensor (top band, 59.4 kDa), **hydolytically cleaved biosensor (middle band, 40.3 kDa), and cleaved biosensor (bottom band, 32.5 kDa). Copy "A" product is not detected due to lack of a His-tag. Note, two gels were used for this experiment as indicated. (D) Velocity profile for B4, B8, and B9 in the presence of different serine protease family members, relative to matriptase activity. Experiments performed in triplicate and reports mean values with standard deviation. Data is normalized for background conditions of biosensor without matriptase.

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. (A) Illustrated schematic of the matriptase mediated activation of Pro-HGF and inhibition by HAI-1. (B) 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

Summary. Sort summary of FACS plots and associated gating for the (A) KDl Round 1 Library and (B) KDl Round 2 Library. See text for sorting strategy methods used.

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). (D) WT KDl and KDl variant matriptase binding normalized to yeast expression, in the presence of increasing concentration of competitive matriptase substrate. * Expression and binding fluorescence is measured using FACS and is quantified from averaging the fluorescent output from at least 10,000 yeast cells/sample. All graphs were plotted and analyzed using GraphPad Prims software and are representative of at least three independent experiments performed. Error bars represent experiments performed in duplicate.

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.

Matriptase inhibition characterization of the wild-type KDl Fc fusion (A) and KDl variant (B) engineered inhibitors plotted (i) with or (ii) without KDl, N54Y. Values are reported as concentration of inhibitor required to reduce matriptase cleavage of a matriptase sensitive biosensor "B4" by 50% (IC50). *Results report mean IC50 with standard deviation values.

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. Each scatter plot represents 10,000 live yeast interrogated for their ability to bind antibodies that recognize yeast expression tags (Hemagglutinin) or WT HAI- 1 specific epitopes. *Fold profiles may vary due to the polyclonal nature of the HAI-1 primary antibody. 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.

DEFINITIONS

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.

Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, 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.

The headings provided herein are not limitations of the various aspects or embodiments of the invention. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with the general meaning of many of the terms used herein. Still, certain terms are defined below for the sake of clarity and ease of reference. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely", "only" and the like in connection with the recitation of claim elements, or the use of a "negative" limitation.

As used herein, the term "malignancy" 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). By "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.

As used herein, the term "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.

As used herein, the term "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.

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. For purposes of this disclosure, a therapeutically effective dose of a compound and/or composition (e.g., compositions that include the compound) is an amount that is sufficient, when administered to the individual, to palliate, ameliorate, stabilize, reverse, prevent, slow or delay the progression of the disease state (e.g., cancer, tumor, etc.). 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. In some embodiments, 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). "Affinity" refers to the strength of binding, increased binding affinity being correlated with a lower KD.

As used herein, the terms "treatment," "treating," and the like, refer 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," as used herein, 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.

As used herein, the terms "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. Thus "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. The term "amino acid" encompasses a- and β-amino acids.

In general, 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. In certain embodiments, peptides are between, 3 and 5 or 5 and 30 amino acids in length. In certain embodiments, a peptide may be three or four amino acids in length.

As used herein, the term "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. The term 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, β- galactosidase, luciferase, etc., and the like.

As used herein, the term "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.

As used herein, the term "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. As used herein, the term "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.

As used herein, the term "assessing" includes any form of measurement, and includes determining if an element is present or not. The terms "determining", "measuring",

"evaluating", "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.

The term "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. In certain cases, 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. The term 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, β- 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.

In the context of a composition, the term "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. For example, 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.

As used herein, 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. Activation of the matriptase zymogen requires sequential N-terminal cleavage, activation site autocleavage, and transient association with HAI-1. 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).

As used herein, the term "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.

As used herein, the term "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).

As used herein, the term "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.

Commun. 2017 73:45-50).

As used herein, the term "multimerization domain" refers to a domain that can be placed into another protein to make that protein multimerize, e.g., dimerize.

Other definitions of terms may appear throughout the specification.

DETAILED DESCRIPTION OF THE INVENTION

Before the present invention is described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a

contradiction.

It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a nucleic acid" includes a plurality of such nucleic acids and reference to "the 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. I-IV), Using Antibodies: A Laboratory Manual, Cells: A Laboratory Manual, PCR Primer: A Laboratory Manual, and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press), Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, New York, Gait, "Oligonucleotide Synthesis: A Practical Approach" 1984, IRL Press, London, Nelson and Cox (2000), Lehninger, A., Principles of Biochemistry 3 ^rd Ed., W. H. Freeman Pub., New York, N.Y. and Berg et al. (2002) Biochemistry, 5 ^th Ed., W. H. Freeman Pub., New York, N.Y., all of which are herein incorporated in their entirety by reference for all purposes.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. Provided herein is a 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. For example, 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. Additionally, residues flanking the C-terminal cysteine (R301,G302,V303) 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.

Using the 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. However, 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

(250,259,275,283,296,299). 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;

particularly when expressed within the context of the HAI-1 protein. However, previous efforts to engineer the KD1 domain directly recovered consensus mutations at residue sites: S253, N254, S277, L284, and 1297 (Figs. 14 and 15). Interestingly, S277 and L284 appear to occur within the secondary structure regions, while S253 and N254 reside in the KD2 region of KD2/1, and therefore suggest that non-deleterious mutations can be made at these sites. Aside from these listed regions, the following loop regions may potentially be targeted and conducive to mutational changes (D269 to 1274; K287 to N288; Y290 to L291). Therefore, it is predicted that non-deleterious mutations can be applied to the existing KD2/1 chimera sequence, at non-critical regions, to explore further functional improvements.

In some embodiments, the polypeptide may further comprise a second Kunitz domain, e.g., another Kunitz domain that is capable of inhibiting matriptase. In some embodiments, 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.

In some embodiments, 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). As noted above, use of a human sequence may be less immunogenic as a therapeutic.

In some embodiments, 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. In some embodiments, 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.

The 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.

( SEQ I D NO : 17 )

In these embodiments, the polypeptide may contain a sequence that is the KD2/1 domain as shown above, and the italicized sequence is an Fc domain. As such, in some embodiments, 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

98% identical, at least 99% identical or 100% identical to 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. 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. For example, the linker may be a flexible linker composed of three or more Gly and/or Ser residues.

In some embodiments, the polypeptide may be combined with a pharmaceutically acceptable excipient. Examples of pharmaceutically acceptable excipient are described below.

A method for inhibiting a matriptase protease is also provided. In some

embodiments, 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. As would be apparent, 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). In some embodiments, the matriptase protease is tethered to the surface of a cell, e.g., a mammalian cell.

Also provided herein is a fusion protein that can act as a biosensor for a protease. In these embodiments, 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. In this fusion 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. In these embodiments, fusion protein emits an optically- detectable signal (e.g., luminescence or fluorescence) when it is in its uncleaved form.

Cleavage of the fusion protein by the protease abolishes the optically-detectable signal. In some embodiments, the reporter protein is a fluorescent protein and the optically detectable signal is fluorescence.

In these embodiments, 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. In some embodiments, the cleavage site may have the amino acid sequence of any of the cleavage sites described herein, e.g., the B4 amino acid sequence. As would be apparent, the N- and C-terminal domains of the fustion protein do not contain any cleavage sites for the protease.

Also provided is 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. In some embodiments, 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). In some embodiments, 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. In these embodiments, 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. In these embodiments, 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. In some embodiments, the cells are yeast cells that display the candidate inhibitors, although other cell types can be used.

Methods of treatment

Also provided is a method of treating a subject for a matriptase-related disease or condition. 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. Because matriptase has been implemented in a variety of different cancers, it is expected that the present polypeptide will be an effective therapy for a variety of cancers including, but not limited to, Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Adrenocortical Carcinoma, AIDS-Related Cancers (e.g., Kaposi Sarcoma, Lymphoma, etc.), Anal Cancer, Appendix Cancer, Astrocytomas, Atypical Teratoid/Rhabdoid Tumor, 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, Craniopharyngioma, Ependymoma, etc.), Breast Cancer (e.g., female breast cancer, male breast cancer, childhood breast cancer, etc.), Bronchial Tumors, Burkitt Lymphoma, Carcinoid Tumor (e.g., Childhood, Gastrointestinal, etc.), Carcinoma of Unknown Primary, Cardiac (Heart) Tumors, Central Nervous System (e.g., Atypical Teratoid/Rhabdoid Tumor, Embryonal Tumors, Germ Cell Tumor,

Lymphoma, etc.), 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,

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,

Gastrointestinal Stromal Tumors (GIST), 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 (Primary), Lobular Carcinoma In Situ (LCIS), Lung Cancer (e.g., Non-Small Cell, Small Cell, etc.), Lymphoma (e.g., AIDS-Related, Burkitt, Cutaneous T-Cell, Hodgkin, Non-Hodgkin, Primary Central Nervous System (CNS), etc.), Macroglobulinemia (e.g., Waldenstrom, etc.), Male Breast Cancer, Malignant Fibrous Histiocytoma of Bone and Osteosarcoma,

Melanoma, Merkel Cell Carcinoma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Midline Tract Carcinoma Involving NUT Gene, Mouth Cancer, Multiple Endocrine Neoplasia Syndromes, Multiple Myeloma/Plasma Cell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes, Myelodysplastic/Myeloproliferative Neoplasms, Myelogenous Leukemia (e.g., Chronic (CML), etc.), Myeloid Leukemia (e.g., Acute (AML), etc.), Myeloproliferative Neoplasms (e.g., Chronic, etc.), Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Non-Small Cell Lung Cancer, Oral Cancer, Oral Cavity Cancer (e.g., Lip, etc.), Oropharyngeal Cancer, Osteosarcoma and Malignant Fibrous Histiocytoma of Bone, Ovarian Cancer (e.g.,

Epithelial, Germ Cell Tumor, Low Malignant Potential Tumor, etc.), Pancreatic Cancer, Pancreatic Neuroendocrine Tumors (Islet Cell Tumors), Papillomatosis, Paraganglioma, Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer, Pharyngeal Cancer, Pheochromocytoma, Pituitary Tumor, Pleuropulmonary Blastoma, Primary Central Nervous System (CNS) Lymphoma, Prostate Cancer, Rectal Cancer, Renal Cell (Kidney) Cancer, Renal Pelvis and Ureter, Transitional Cell Cancer, Retinoblastoma,

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

Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Cell Carcinoma, Squamous Neck Cancer (e.g., with Occult Primary, Metastatic, etc.), Stomach (Gastric) Cancer, T-Cell Lymphoma, Testicular Cancer, Throat Cancer, Thymoma and Thymic Carcinoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Ureter and Renal Pelvis Cancer, Urethral Cancer, Uterine Cancer (e.g., Endometrial, etc.), Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer, Waldenstrom Macroglobulinemia, Wilms Tumor, and the like. In some instances 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, small cell carcinoma , small cell lung carcinoma, spindle cell carcinoma, squamous cell carcinoma, terminal duct carcinoma, transitional cell carcinoma, tubular carcinoma, verrucous carcinoma, and the like. In some cases, the individual has recently undergone treatment for cancer (e.g., chemotherapy, radiation therapy, etc.) and are therefore at risk for recurrence. Any and all cancers are suitable cancers to be treated by the subject methods, compositions, and kits.

In some embodiment, the polypeptide may inhibit cancer progression, e.g., cancer metastasis, in the subject.

Formulations

In the subject methods, a subject polypeptide can be administered to the host using any convenient means capable of resulting in the desired therapeutic effect or diagnostic effect. Thus, the agent can be incorporated into a variety of formulations for therapeutic administration. More particularly, a subject polypeptide can be formulated into

pharmaceutical 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.

In pharmaceutical dosage forms, a subject polypeptide 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.

For oral preparations, 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.

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 gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N- methylglucosamine, galactosamine, and neuraminic acid; and/or non-ionic surfactants such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).

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. Examples of 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. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term "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). Examples of suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). Examples of suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Examples of suitable Polyoxyethylene alkyl ethers are those sold under the trademark Brij™.

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. For example, known 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.

In some embodiments, 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. In other embodiments, a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% (w/v).

For example, 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.

As another example, 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.

As another example, 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; 0.02% Tween 20 w/v; 20 mM L-histidine; and 250 mM trehalose; and has a pH of 5.5.

As another example, 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 subject polypeptide; 0.01% Tween 20 w/v; 10 mM L-histidine; and 125 mM trehalose; and has a pH of 5.5; or 6) 5 mg/mL of a subject polypeptide; 0.02% Tween 20 w/v; 20 mM L-histidine; and 250 mM trehalose; and has a pH of 5.5; or 7) 75 mg/mL of a subject polypeptide; 0.02% Tween 20 w/v; 20 mM L-histidine; and 250 mM mannitol; and has a pH of 5.5; or 8) 75 mg/mL of a subject polypeptide; 0.02% Tween 20 w/v; 20 mM L histidine; and 140 mM sodium chloride; and has a pH of 5.5;or 9) 150 mg/mL of a subject polypeptide; 0.02% Tween 20 w/v; 20 mM L-histidine; and 250 mM trehalose; and has a pH of 5.5; or 10) 150 mg/mL of a subject polypeptide; 0.02% Tween 20 w/v; 20 mM L- histidine; and 250 mM mannitol; and has a pH of 5.5; or 11) 150 mg/mL of a subject polypeptide; 0.02% Tween 20 w/v; 20 mM L-histidine; and 140 mM sodium chloride; and has a pH of 5.5; or 12) 10 mg/mL of a subject polypeptide; 0.01% Tween 20 w/v; 20 mM L- histidine; and 40 mM sodium chloride; and has a pH of 5.5. A subject polypeptide can be utilized in aerosol formulation to be administered via inhalation. A subject polypeptide can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.

Furthermore, a subject polypeptide can be made into suppositories by mixing with a variety of 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. Similarly, 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.

The term "unit dosage form," as used herein, 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.

Other modes of administration will also find use with the subject invention. For instance, a subject polypeptide can be formulated in suppositories and, in some cases, aerosol and intranasal compositions. For suppositories, the vehicle composition will include traditional binders and carriers such as, polyalkylene glycols, or triglycerides. Such 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. Typically, 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. In addition, if desired, the vehicle may contain minor amounts of 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. Moreover, 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.

In some embodiments, 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.).

The various 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.

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.).

There are a number of 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.).

Dosages 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 μg to 10 mg per kilogram of body weight per minute.

Those of skill will readily appreciate that 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.

Routes of administration

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.

Conventional and pharmaceutically acceptable routes of administration include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, topical application, intravenous, intraarterial, rectal, nasal, oral, and other enteral and parenteral routes of administration. Routes of administration may be combined, if desired, or adjusted depending upon the polypeptide and/or the desired effect. 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. In general, 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

administration other than through the alimentary canal. 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.

By 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. As such, treatment 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.

terminated, such that the host no longer suffers from the pathological condition, or at least the symptoms that characterize the pathological condition.

In some embodiments, 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.

A variety of hosts (wherein the term "host" is used interchangeably herein with the terms "subject," "individual," and "patient") are treatable according to the subject methods. Generally such 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). In some embodiments, the hosts will be humans.

Kits with unit doses of a subject antibody, e.g. in oral or injectable doses, are provided. In such 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. EMBODIMENTS

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).

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:

contacting the matriptase protease with a polypeptide of any of embodiments 1-9, thereby inhibiting the matriptase protease.

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).

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:

(a) a N-terminal domain of a reporter protein; (b) a cleavage site for a protease; and (c) a C-terminal domain of the reporter protein, wherein the fusion protein emits an optically-detectable signal and wherein cleavage of the fusion protein by the protease abolishes the optically-detectable signal.

Embodiment 22. The fusion protein of embodiment 21, wherein reporter protein is a fluorescent protein and the optically detectable signal is fluorescence Embodiment 23. The fusion partner of any of embodiments 21-22, wherein the protease is matriptase.

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. EXAMPLES

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

In this example, 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 final protein product contains 4 stoichiometric binding sites that effectively inhibit matriptase with a Ki =70 + 5 pM, an increase of 120-fold compared to the natural HAI-1 inhibitor, this make it one of the most potent matriptase inhibitors identified to date. Furthermore, 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. EXAMPLE 1

HIGH AFFINITY ENGINEERED MATRIPTASE INHIBITOR Methods for Example 1

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. Following washing, 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 μΜ; 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 μΜ 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 . Relative velocities were plotted against inhibitor concentration, and apparent inhibition constants, Ki ^app , values were determined by fitting each curve to the Morrison binding equation (1) as previously described (38, 40, 58) using GraphPad Prism version 6 software. Inhibition constants, Ki, were then calculated using equation (2).

Media and reagents. 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. 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

transformation, serially diluted, and plated on SD-CAA plates to quantify a transformation efficiency of 5xl0 ⁷ cells. Cells were then expanded in SD-CAA media at 30°C and 5xl0 ⁸ cells were induced for surface expression in SG-CAA media at 20°C in preparation for library sorting. Additional rounds of mutagenesis followed the same method as above (46), but the concentration of Mn ²⁺ was increased to 0.3 mM or 0.4 mM to increase the mutational frequency. It appears the wild-type KD1 gene (Cys250 to Val303; Genbank Accession ID: AY358969.1) was present during PCR and transformation, allowing recombination of genetic regions of KD1 and KD2 to generate clone 33. Final PCR products were then amplified as before and transformed into EBY100 with an estimated transformation efficiency of lxlO ⁷ cells. Yeast cells were expanded and induced for expression prior to sorting as described above.

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). 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).

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 μΐ 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 μΐ 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 μΜ 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). The reactions were incubated at 37°C, 5% CO2 between reads. The obtained matriptase activity rates, RFU/hr, were then normalized to conditions lacking inhibitor, and plotted against inhibitor concentration tested. Graphs were fitted using a three-parameter equation (GraphPad version 6) and IC50 values were calculated.

Cancer cell invasion assay. 24-well 8μιη pore inserts (Corning) were coated with 50 μg of Matrigel (Corning) following manufacturing protocols and inserted into a 24-well plate. lxlO ⁴ cancer cells in 0.5 mL 2% serum- supplemented media were seeded onto the Matrigel coated inserts and lxlO ⁴ human embryonic kidney (HEK) cells in 0.75 mL 2% serum- supplemented media were then seeded onto the lower chamber; 100 nM of soluble inhibitor was added to the lower chamber immediately. The 24-well plates were cultured for 48 hr in a humidified tissue culture incubator at 37°C, 5% CO2 atmosphere. Following incubation, non-invading cells were removed and invaded cells were stained using crystal violet then imaged at three random center fields of view at lOx magnification. Images were analyzed using Image J, adjusted for brightness-contrast and cell borders defined by watershed command, and cell numbers were quantified with threshold particle setting <500 pixels excluded to reduce artifacts.

Recombinant protein production. 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). Genes were cloned into the pCEP4 vector using Notl and Hindlll restriction sites, and included a C-terminal hexa- histidine tag (Pro-HGF, monomer inhibitors) or a mouse IgG2a Fc domain was genetically linked using 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 using 400 μg/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, followed by culturing for at least one week. 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.

The 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.

(SEQ ID NO : 20 )

Results for Example 1

Engineering the Kunitz domain 2 (KD2) of HAI-1 to bind matriptase

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). In that work, removal of the KD2 domain resulted in a 10-fold improvement in HAI- 1 inhibition of matriptase activity. In addition, matriptase inhibition was proposed to be driven by a 4 amino acid primary binding interface (Arg-Cys-Arg-Gly) found in KDl, but absent in KD2 (Fig. 1 and Fig. 5 A). To create an improved matriptase inhibitor, the inactive KD2 domain of HAI- 1 was targeted for conversion into a matriptase binding module, effectively doubling the binding sites within the HAI-1 protein.

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). In an attempt to supplement this binding site, primary matriptase binding site residues (Arg-Cys-Arg: KD2-graft 1, or Arg-Cys-Arg-Gly: KD2- graft 2) were introduced into KD2 from KDl (Fig. 5 A). Yeast-displayed versions of these constructs revealed high surface expression but a lack of matriptase binding similar to wild- type KD2 (Fig. 5 D). These results indicated further engineering was required to effectively convert KD2 into a matriptase binding domain.

To further explore additional mutation space beyond the grafted primary binding motif, error-prone polymerase chain reaction (epPCR) (46) was applied to randomly introduce mutations throughout the KD2-graft 2 gene. The mutated DNA was transformed into S. cerevisiae yeast cells, resulting in ~5xl0 ⁷ transformants which were induced to express a library of yeast- surfaced displayed KD2 variants, averaging 2 amino acid mutations per gene. The library was screened using fluorescence-activated cell sorting (FACS) to isolate yeast clones that expressed KD2 variants and also bound to matriptase (Fig. 2A). Yeast cells that were collected were grown in culture and induced for KD2 expression for additional screening. Following two rounds of library screening, DNA was recovered from the pooled yeast and subject to epPCR to introduce additional genetic diversity, with an average mutagenic frequency of 2 to 5 amino acids per gene. A new library of -lxlO ⁷ yeast transformants was created and screened by FACS to identify KD2 variants that bound to matriptase. Parallel sorting and analysis for non-specific binding to secondary antibodies was also performed to reduce occurrence of false positive binding variants. From these efforts, a yeast population emerged that demonstrated significantly improved binding to matriptase compared with wild-type KD2 (Fig. 2 A).

Characterization of sorted library variants reveal a KD2/KD1 chimera

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). Surprisingly, 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). An equilibrium binding assay showed that yeast-displayed KD2/1 binds to matriptase with an affinity of K _d = 220 + 30 pM (Fig. 2 C), which was 20-fold weaker than KDl. Notably, inhibitors that demonstrate protease binding do not always demonstrate protease inhibition of the target active site (47, 48). Thus, proteins were tested for functional matriptase inhibition by incubating an increasing number of yeast cells displaying KD2/1 or wild-type KDl or KD2 with soluble matriptase and substrate. Fluorescent matriptase substrate activation was quantified over time and revealed that both yeast-displayed KDl and KD2/1 domains significantly inhibited matriptase activity in a cell number dependent manner, while the yeast-displayed KD2 domain did not inhibit matriptase, even with up to 10 ⁴ yeast cells (Fig. 2 D).

Development of a soluble matriptase inhibitor

After validating that yeast-tethered KD2/1 could bind to and inhibit matriptase, 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). In addition, given the favorable binding properties of yeast- displayed KDl (Fig. 2 C) an alternative HAI-1 design was created where the wild-type KD2 domain was replaced with a second wild-type KDl domain, termed 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). Thus, 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. 3; HAI- R260A-Fc; KD1-R260A-KD2/1-Fc). Each protein construct was expressed in a transient mammalian cell expression system and underwent two-step purification. The resulting size exclusion chromatograms demonstrate expression and purification of all proteins (Fig. 7 A- B), with the exception of KDlx2-Fc (Fig. 7 C-D). These findings suggest that the 6 N- terminal amino acids from wild-type KD2, found in the KD2/1 chimera (Fig. 12), were critical for effective protein folding/ expression in the context of full-length HAI-1.

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. As expected, 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. In contrast, 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 HAI-1 monomer has a moderate Ki of 9.1 + 1 nM, which improves slightly by 2-fold as a bivalent HAI-1 Fc fusion (Ki = 4.2 + 0.5 nM). Additionally, 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:

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.

Finally, 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

improvement in Ki of 4-fold compared with the wild- type KD1 monomer, a 60-fold improvement compared with HAI-Fc, and a 120-fold improvement compared with the HAI- 1 monomer. The increased potency is due to the replacement of the KD2 domain with the matriptase binding KD2/1 chimera, as well as the homodimeric nature of the Fc fusion construct. These combined engineering efforts expand the number of matriptase binding sites from one domain in wild-type HAI-1 to four domains within the final KD1-KD2/1-Fc construct. Further experiments also determined that KD1-KD2/1-Fc follows a competitive inhibition modality for matriptase (Fig. 9) similar to that of wild-type KD1 monomer (26).

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). In contrast, 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. Additionally, 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. Overall, the relative selectivity of 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

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). 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 After demonstrating potent and selective inhibition of the in vitro form of matriptase, 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). To stimulate cancer invasion, 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. A significant increase in invasion of both breast (MDA-MB-231) and lung (A549) cancer cells was observed upon incubation with HEK-huPro-HGF cells (Fig. 4 B), compared with controls of cancer cells alone or with untransfected HEK cells. Furthermore, when KD1-KD2/1-Fc was added to the reaction containing HEK-huPro-HGF cells, the invaded cell number decreased significantly compared to conditions without inhibitor.

Collectively, these results indicate that 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.

Discussion for Example 1

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). 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

recombinantly expressed in mammalian cell culture.

Notably, the 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. Results obtained with the KD1-R260A-KD2/1-Fc inhibitor support this possibility, in which the KD2/1 domains functionally inhibit matriptase within the context of the Fc fusion construct with a Ki= 550 + 50 pM. It is important to note that although non- inhibitor depleting conditions were used to quantify the Ki value for all inhibitors tested, Ki values may be an overestimate, as the concentration of matriptase used (50 pM) was at the lowest limit for assay detection and may also not be 100% active under the assay conditions. Further testing using solid phase or solution based assay formats could help confirm the Ki for this very tight matriptase inhibitor, assuming improved detection limits. However, the Ki of wild-type KDl monomer that is reported (310 + 20 pM) closely aligns with prior literature values for rat (328 + 181 pM) and human (380 + 70 pM) matriptase, which use similar assay methods and thus strongly validates these results (34, 42).

KD1-KD2/1-FC additionally retains the highest selectivity to matriptase amongst a panel of serine proteases tested (Table 2). Interestingly, while 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. Like matriptase, the relative fold difference in Ki values for hepsin between KD1-KD2/1-Fc and wild-type KDl monomer is also 4-fold, which further supports the stoichiometric inhibition hypothesis stated above. Hepsin is reported to share a similar role as matriptase in cancer progression, and thus dual targeting of hepsin and matriptase by KD1-KD2/1-Fc may serve as an attractive therapeutic feature (28, 29, 35).

Table 2:

Table 2: 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.

Cell based activity assays further demonstrate the superior potency of KD1-KD2/1- Fc in inhibiting matriptase activity both soluble and cell associated form. The huPro-HGF activation assay qualitatively confirmed that 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). The greater magnitude of improvement in cancer cell matriptase inhibition (10-fold) compared to soluble matriptase (4-fold) is possibly due to avidity effects of the KD1-KD2/1-Fc construct on cell anchored matriptase. 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). Notably, 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. This high background of protease activity, combined with the heterogeneity of other serum proteins and constitutively overexpressed huPro-HGF, likely contributes to preventing a greater reduction in cancer cell invasion observed with KD1-KD2/1-Fc. Further testing using optimized media conditions might result in a greater extent of matriptase inhibition in cancer cells upon treatment with KD1-KD2/1-FC.

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. In addition to wild-type KD1 (Ki = 310 pM + 20 pM) (42, 53), previous efforts have generated matriptase inhibitors based on constrained peptides (Ki = 830 + 140 pM (40) and Ki = 290 + 54 pM) (38) and antibodies (Ki = 720 pM) (39). Peptide and KD1 -based inhibitors, as well as synthetic small molecule inhibitors (36, 37), have short circulating half-lives and restricted molecular surface area for binding matriptase with high affinity. The engineered KD1-KD2/1-FC protein fulfills several attractive design criteria. First, use of the native HAI- 1 as a starting point for therapeutic development leverages the affinity and specificity of the natural inhibitor. Second, replacing the KD2 domain with an active matriptase-binding domain is expected to be minimally perturbing to native HAI-1. Third, 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. Fourth, 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.

Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims.

References for Example 1

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2. Ye J, et al. (2014) Loss of hepatocyte growth factor activator inhibitor type 1 participates in metastatic spreading of human pancreatic cancer cells in a mouse orthotopic transplantation model. Cancer Sci 105:44-51.

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doi: 10.1002/9781118540398.

EXAMPLE 2

SCREENING METHOD

Methods for Example 2

Media and reagents: 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 μΜ 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.

Protease selectivity screen: 1 μΜ 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 μΜ) 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-

Western Blot Qualitative Cleavage Analysis: 1 μΜ of each biosensor was added to 10 nM recombinant human matriptase and kinetically monitored for fluorescent output overnight as described above. Each biosensor without matriptase (buffer only) served as a non-cleaved control. The reaction products, along with matriptase alone or buffer alone controls, were first denatured by boiling at 95°C for 10 min, then loaded onto a 12% polyacrylamide gel (GenScript) and subjected to electrophoresis for protein fragment separation. Following SDS-PAGE, 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. After 3, 5 minute wash steps in IxTBST, membranes were transferred to IxTBST containing 5% dry milk and (1 : 10,000) dilution anti-rabbit horse radish peroxidase (HRP) secondary antibody (Fisher Scientific) for one hour at room temperature. Membranes were again washed as above and protein bands were then detected using SuperSignal West Femto HRP substrate (Fisher Scientific). Soluble Inhibitor Production: DNA encoding the open reading frame of the human KD1 domain of the HAI-1 protein (amino acids Cys250 to Val303; Genbank Accession ID: AY358969.1) was cloned into the pPIC9 yeast expression plasmid, amplified using DH10B electrocompetent E. coli cells, and confirmed by sequencing. Methods of pPIC9 cloning, transformation into P. Pastoris, and protein expression was completed using reagents, media, and protocols exactly as previously described. ³⁷ The KD1 protein was expressed in P. Pastoris, and purified by Ni-NTA metal chelating chromatography and size exclusion chromatography. Purified protein was characterized using polyacrylamide gel

electrophoresis (PAGE) and 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).

Yeast Display of Matriptase Inhibitor: 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

(Invitrogen) and a 1: 100 dilution of anti-rabbit fluorescein isothiocyanate-FITC (Abeam) for 15 minutes at 4°C. Yeast were then washed with assay buffer and fluorescent antibody binding was measured using flow cytometry (BD Accuri) and data analyzed (BD CSampler software).

Matriptase binding to yeast-displayed KD1 proteins was carried out by first combining 10 ⁵ yeast (OD = 10 ⁷ yeast/mL) with 10 nM human matriptase using

manufacturer's buffer conditions, followed by incubation for 24 h at room temperature. Samples were then washed and stained for yeast expression (as above), and matriptase binding (1 : 100 dilution of anti-His FITC (Bethyl)). Samples were washed and maintained on ice until loading onto a flow cytometer for analysis as described above.

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 μΜ 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. Conditions of B4 with matriptase alone or with buffer alone, with or without non-induced yeast, served as control 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.

Cancer Cell Matriptase Expression: 5 x 10 ⁵ cancer cells were resuspended in cold

IxPBS with 1 mg/mL bovine serum albumin (0.1% BPBS) solution, containing a 1: 100 dilution of mouse anti-human matriptase antibody (Fisher Scientific). 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.

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. ΙμΜ 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.

Results/Discussion for Example 2

To create a ddRFP-based, matriptase sensitive biosensor, a series of amino acid linkers that could be recognized and cleaved by matriptase were designed. A panel of linker designs was created based upon the sequence of a natural matriptase substrate, called pro- macrophage stimulating protein (pro-MSP). Pro-MSP is an inactive growth factor, that upon cleavage by matriptase, is converted into active MSP which binds its cognate receptor, RON, to stimulate downstream cellular events including growth and proliferation. ¹⁶ 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. ²⁷ To develop a matriptase cleavable linker within the context of ddRFP, 8 linkers of increasing lengths, each derived from the natural cleavage sequence of MSP, were designed. Each design was named for the number of amino acids flanking each side of the scissile bond (i.e. B3 with three flanking residues, up to BIO with ten flanking residues) (Fig. 16 C).

Following E. coli production and soluble purification, 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. For biosensors exhibiting ideal behavior, fluorescence decreases over time in the presence of matriptase, but is retained over time in the absence of matriptase. Interestingly, while constructs B4 through B9 portray these ideal fluorescent properties, B3 exhibits no change in fluorescence over time with matriptase, while BIO reveals low fluorescence. To further explore this trend, the change in each biosensor design' s initial velocity in response to increasing matriptase concentration to determine the effects of linker length on matriptase activity was examined (Fig. 17 B). Once again, B3 shows low velocity of matriptase cleavage, while B IO exhibits essentially no change in fluorescence even with the addition of 10 nM matriptase. In contrast, the velocity of matriptase activity increases with increasing linker length for constructs B4 to B9. These results help support the presence of linker specific cleavage events by matriptase, as opposed to off target cleavage of the A or B fluorescent protein domains, in which all biosensor designs would show the same velocity profiles, including B3. Additionally, this trend was observed for conditions of both 10 nM and 1 nM matriptase, with a corresponding loss in velocity at lower concentration, suggesting a specific response to matriptase concentration and loss of fluorescence due to cleavage by matriptase.

To further investigate the effects of a linker-specific cleavage event by matriptase, the reaction products of each biosensor design, incubated with or without 10 nM matriptase, were analyzed. The analysis used SDS-PAGE followed by Western blot. Western blot analysis enables detection of the hexahistidine tag of each biosensor and its matriptase cleaved products. These results reveal at most three bands for each construct (Fig. 17 C): the uncleaved biosensor substrate (59.4 kDa), the matriptase cleaved biosensor product (32.5 kDa), and the hydroly tic ally cleaved biosensor product (40.3k Da); the latter of which is produced from boiling each protein for denaturation. ^{26 28} Western blot analysis reveals that no matriptase cleavage product is observed for the B3 linker design, confirming its lack of matriptase susceptibility and supporting the prior velocity data (Fig. 17 A-B). It is likely that the B3 linker is too short and thus sterically inaccessible for matriptase recognition and cleavage within the context of the ddRFP construct. In contrast, matriptase cleaved products are observed for B4 to B9, in agreement with the previous velocity data and confirming linker-specific cleavage by matriptase. Interestingly, BIO also reveals a cleavage product which indicates that the linker region is present and accessible in this construct. The diminished fluorescent signal of the intact B IO protein may be the result of misfolding or disruption of the A-B dimer interface, which would impair ddRFP stabilizing interactions and thus fluorescent output. Finally, the B5 and B6 constructs have cleavage products in lanes lacking matriptase (Fig. 17 C). This finding may also be observed in time course data for the B5 and B6 constructs (Fig. 17 A) in which precipitous loss in fluorescence is observed and may indicate instability or degradation of these biosensor designs. Overall, the kinetic and western blot results confirm linker- specific cleavage by matriptase and reveals poor functional characteristics of the B3, B5, B6, and BIO designs.

For the remaining biosensor designs (B4, B8, and B9), the effects of linker length on matriptase selectivity were tested. Target selectivity is an important consideration in protease biosensor design for measuring protease activity in a heterogeneous environment. To test selectivity, 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). Interestingly, minimal activity is observed for trypsin 3, urokinase, and hepsin, while the velocity of kallikrein 4 increases with increasing linker length, approaching the velocity of matriptase activity. These results indicate that a longer linker between the A and B domains allows for increased promiscuity and recognition by other serine proteases. From these collective findings, further characterization of the B4 construct was pursued due to its superior stability, ideal kinetic properties, and matriptase selectivity. Additional studies revealed that 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. Matriptase expression and functional activity was tested using a human matriptase specific antibody and the commercially matriptase substrate (Boc-QAR-AMC, R&D Systems), respectively; which appeared to strongly correlate for each cell line tested. Next, the ability of B4 to detect matriptase activity expressed on human cancer cell lines was tested (Fig. 18 A) and confirmed significant B4 processing in the presence of cancer cell lines compared with media only control. It is important to note that the heterogeneous environment of cancer cells may contribute additional proteases or other factors that may aid in B4 degradation. However, the distribution of matriptase activity measured by B4 for each human cancer cell line appears to correlate with the distribution measured by the matriptase specific antibody and commercial substrate, suggesting that B4 cleavage was the result of cell associated matriptase activity.

To measure matriptase-inhibited cleavage of B4, an inhibitor domain derived from the matriptase hepatocyte growth factor activator inhibitor type-1 (HAI-1) was used. HAI-1 naturally regulates matriptase activity in normal healthy tissue and is primarily expressed on the surface of epithelial cells to block the substrate-activating properties of matriptase. The multi-domain structure of HAI-1 includes the extracellular Kunitz domain 1 (KDl), which has been previously established to be the main inhibitory domain of HAI-1. ^{33 34} To test inhibition of B4 activation, KDl was recombinantly expressed and purified as a soluble protein. Following confirmation that soluble KDl can inhibit matriptase using the Boc- QAR-AMC substrate as a positive control, 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.

It was shown that the 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 . However, most protease screening methods rely on inhibitor-protease binding, rather than functional inhibition as a criteria for selection. By demonstrating that 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. ³⁶ An inactive KD1 variant was displayed by introducing an point mutation at positon 260 (KD1-R260A) that disrupts matriptase binding. ³⁴ 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. In addition, 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.

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EXAMPLE 3 Materials and Methods for Example 3

Media and reagents: 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. 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.

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

Scientific)) was determined for each construct and measured using flow cytometry (BD Accuri).

KDl Library; Round 1 was constructed using error-prone PCR (epPCR) as previously reported [43]. In short, variable concentration of Mn ²⁺ (0.075mM and 0.15mM) was added to reactions to increase mutational frequency, while elevated ratios of dCTP and dTTP nucleotides were added in order to account for mutational bias. 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' : tgtcagttcctgcaagtcttctt (SEQ ID NO: 19). Final PCR products were amplified in 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 [44]. Cell samples were collected post transformation and serially diluted and platted on SD-CAA plates to quantify a

transformation efficiency. Cells were then expanded in SD-CAA media at 30°C and induced for surface expression in SG-CAA media at 20°C in preparation for library sorting. KDl Library; Round 2 mutagenesis followed the same method as above [43], but utilized the KDl Library; Round 1 ; Sort 5 and 6 as template DNA and the concentration of Mn ²⁺ (0.3mM and 0.4mM) in order increase the mutational frequency. Final PCR products were then amplified as before and transformed into EBY100 as described [44]. Yeast cells were then expanded and induced for sorting as mentioned.

Yeast surface display library screening conditions: Transfromed EBY100 yeast cells were counted, washed, and then mixed with soluble matriptase (final concentration 0 - ΙΟηΜ) 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 (1:250 dilution of anti-HA, mouse primary antibody (Fisher Scientific)) by incubating reactions at RT for 30min.

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. 3-5) with SG-CAA media at 20°C overnight. Competitive and off rate library sorting conditions included steps mentioned above, however after initial matriptase incubation, yeast cells were washed then incubated with soluble competitor inhibitor or substrate. Reactions were then incubated for up to 42 hours (competing inhibitor/off rate) or up to 1 hour (competing substrate) prior to wash and antibody staining steps before FACS sorting._The final library pool was lysed for DNA extraction (Zymoprep; Fisher Scientific), and transformed into DH10B

electrocompetent E. coli cells for plasmid amplification, and then lysed for DNA extraction (Miniprep; Fisher Scientific) and sequencing.

Soluble protein production: 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 400μg/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. 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.

Matriptase Substrate "Biosensor 4" Production: Following transformation into

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. coli inclusion bodies using Bacterial Protein Extraction Reagent (BPER- Thermo Fisher), purified via Ni-NTA chromatography, and then peak 570nm absorbance values were measured to quantify final yields and concentration using the reported extinction coefficient (48,300M ¹ cm ¹ ) and molecular weight (59.4kDa) [45].

Protease inhibition assay: ΙΟηΜ matriptase (R&D) was first added to soluble inhibitor (final concentrations ranging 0-500nM) containing matriptase assay buffer. ΙμΜ 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]. Additionally, 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 displayed KD1 library. Round 1, developed using random mutagenesis:

In order to introduce new mutations within the wild-type KD1 gene sequence and identify affinity matured variants, it was decided to utilize a technique known as directed evolution by yeast surface display. 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)..After confirming wild-type KD1 domain can be successfully displayed and folded on the surface of yeast and characterized for binding to matriptase (FIG. 28, B-C), the next step involved applying error prone polymerase chain reaction (epPCR) to randomly introduce new mutations throughout the wild-type KD1 gene. This process effectively incorporated diverse mutations throughout the wild-type KD1 gene sequence, with a goal of altering its native protein expression and matriptase binding properties. The DNA library was then transformed into S. cerevisiae yeast cells, which were induced to express the KD1 Library; Round 1, comprised of

1.05xl0 ⁷ unique KD1 variants. 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 (ΙΟΟηΜ HAI-1, 15hr), Sort 5 (300nM HAI-1, 20hr), and Sort 6 (300nM HAI-1, 42hr). In all cases, 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).

Sequencing confirmed consensus mutations recovered from KDl library. Round 1:

Final KDl domain variants were collected and sequenced from the KDl Library; Round 1; Sort 5 and Sort 6 populations. (FIG. 21) reveals that consensus mutations are highly prevalent at positions S253 and N254 as well as 1297 (FIG. 23, A). Interestingly, no mutations are present in the primary binding motif region, R258 to G261, indicating this to be a critical and well optimized interface for matriptase interaction. It is also interesting that although only a single amino acid is prevalent at sites S253 (Proline) and 1297 (Valine), position N254 is comprised of a variety of side chains including: positively charged Lysine, hydrophobic Phenylalanine, and polar Tyrosine. Mapping the N254 mutational spectra and location onto the wild-type KDl -matriptase crystal structure complex reveals that the mutational location is distal from the binding interface (FIG. 23, A), and thus may be more amendable to accepting a greater variety of mutations. The next step was to measure and compare the binding affinity (Kd) of matriptase to yeast displayed wild-type KDl and S253 and N254 single mutant variants using a direct yeast display binding assay and FACS analysis. Results revealed that no significant improvement of matriptase binding affinity was observed for each single KDl mutant vs wild-type KDl (Fig. 24, A). This result could likely be attributed to the limits of binding detection by yeast display, in which distinction of Kd at or below ΙΟρΜ is highly difficult. Therefore it could be likely that the mutations do in fact improve the matriptase binding or stability of KDl, though this improvement is not measurable at this level of high initial affinity.

Yeast displayed KDl library. Round 2, developed using random mutagenesis:

Due to the lack of significant improvement of wild-type KDl matriptase binding or yeast expression, it was decided that the next step would be to perform an additional round of directed evolution upon the KDl variants isolated from KDl Library; Round 1 ; Sort 5 and Sort 6. Library construction was performed by applying epPCR with DNA plasmids isolated and pooled from these sort rounds, with a goal of adding additional mutations to these existing alterations. The DNA library was then transformed into S. cerevisiae yeast cells, which were induced to express the KD1 Library; Round 2, comprised of 1.2 x 10 ⁷ unique KD1 variants. The initial mutation frequency averaged 1-8 amino acid mutations per gene with over 50% of the library folded and expressed (FIG. 20, B). 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 ΙΟηΜ matriptase and isolating cells that retained binding and expression of KD1 variants. Competitive binding sorts (Sort 2-5) involved first incubating cells with InM matriptase, then washing away non-binding matriptase prior to incubation with increasing concentration of soluble B4 substrate; Sort 2 (ΙμΜ), Sort 3 (10μΜ), Sort 4 (20 μΜ), and Sort 5 (30μΜ) (*note, B4 matriptase Km = 8.17 + 1.02 μΜ). In all cases, yeast were analyzed using FACS for yeast displayed KD1 variants that bound to matriptase and were then collected and cultured for repeating sorting rounds of directed evolution (FIG. 29).

Sequencing confirmed consensus mutations recovered from KD1 library. Round 2:

Final KD1 domain variants were collected and sequenced from the KD1 Library; Round 2; Sort 4. (FIG. 22) reveals that once again consensus mutations are highly prevalent at positions S253 and N254, but additional mutations arise at positions S277 and L284. Interestingly, once again no mutations are present in the primary (R258 to G261) or secondary (R265, N286, and N289) binding motif regions, indicating this to be a critical and well optimized interface for matriptase interaction. It is also interesting that although only a single amino acid is again prevalent at site S253 (Proline) as well as S277 (Arginine) and L284 (Methionine), position N254 is still comprised of a variety of side chains; however the variety is centered around only polar (Serine, Tyrosine) and positively charged (Lysine) side chains, now lacking the prior hydrophobic options. Additionally, the consensus mutation C297V from KD1 Library; Round 1 is now missing, suggesting it is no longer necessary for competitive matriptase binding in the present of very high B4 substrate concentrations.

Mapping the new mutations from Round 2 with the N254 mutational spectra from Round 1 onto the wild- type KD1 -matriptase crystal structure complex reveals that the mutational location remains distal from the binding interface while potentially interacting with matriptase allosteric loop regions or providing novel stabilizing interactions internal to KD1 (FIG. 23, A). S277R in particular has the potential of introducing new hydrogen bonds in KD1 to stabilize an alpha helix to help orient the binding interface to matriptase and form a more rigid structure. It is also interesting to note from sequence comparison that S277R mutation occurs independent of N254 mutation. Closer inspection of combining S277R mutation with either N254Y (FIG. 23, B) or 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:

In order to test these hypothesis and measure phenotypic improvements of yeast displayed KD1 variants, the next step involved developing double and single mutant KD1 clones comprised of: N254Y, N254K, S277R, N254Y/S277R, N254K/S277R, and

S253P/L284M. While little improvement in yeast expression was measured for N254Y

(FIG. 24, B), a greater difference in matriptase binding with competitive substrate was observed for some KD1 variants. By first incubating yeast displayed KD1 variants with ΙΟηΜ soluble matriptase, then adding 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. Interestingly, under these assay conditions it appears that all variants have greater IC50 values compared with wild-type KD1, with S277R single mutant achieving the highest IC50 of 14μΜ Biosensor 4 (FIG. 24, C). One concern is that KD1 variants have greater binding values due to higher overall expression levels on yeast. To investigate this question, the matriptase binding to yeast expression, for varying concentrations of B4 competing matriptase substrate was normalized (FIG. 24, D). These results clearly reveal that although the N254Y/S277R variant has poor yeast expression (FIG. 24, B), it has up to a 3-fold improved normalized binding capacity for matriptase compared to wild-type KD1 ; even up to 60μΜ B4. Additionally, the normalized binding capacity is dose dependent on Biosensor 4 concentration, indicating that the matriptase binding interaction is competitive and reversible. It is also interesting that this improvement is not observed for N254Y single mutant variant, suggesting that the combined S277R mutation does in fact improve functional activity when combined with N254Y (FIG. 23, B), possibly by alleviating steric clashes imposed by N254 mutations. KDl mutants demonstrate slightly improved competitive binding to matriptase compared with wild-type KDl:

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). Additionally, it was of interest to design a matriptase activatable inhibitor construct, and replace the Gly4Ser linker with a matriptase cleavable linker (MCL) derived from the B4 matriptase cleavage site: RSKLRVGGH (SEQ ID NO: 21). Each soluble KDl Fc fusion construct was expressed and purified at sufficient levels for testing (FIG. 25. B). In parallel, the N254Y, S277R, or combined N254Y/S277R KDl mutations, which demonstrated improved properties as yeast displayed variants, were chosen for soluble inhibitor construct design. For these designs, 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. Interestingly, 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.

Additionally, the MCL design was also a very poor inhibitor of matriptase, suggesting this design is no longer feasible to outcompete the wild-type KD1 monomer design. Next, the ability of KD1 mutations isolated from directed evolution to improve matriptase inhibition compared to wild-type was tested. Experimental conditions were used as noted above and IC50 values were plotted for select inhibitor constructs (FIG. 27, B ). Although it appears that some improvement is made comparing wild-type inhibitors to variants within each soluble construct category, no improvement is significant. The greatest difference in matriptase inhibition occurs for the HAI-1 "full length" Fc fusion constructs, in which single mutant N254Y or S277R reveal a 3.7nM IC50 and is comparable to the wild-type KD1 monomer (2.28nM).

This result is also interesting that 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). While 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. This sensitivity to regulation may also be the reason why the KD1 Fc fusion constructs lost potency for matriptase inhibition compared with wild-type KD1 monomer, due to proximal domain interactions and loss in folding. In fact, interdomain relationships within the HAI-1 context were also observed in a recent crystal structure publication, in which expression and function was lost due to disrupting interdomain interactions [31]. Alternatively targeting the wild-type KD2 domain for engineering was a more effective strategy for engineering a more potent matriptase inhibiting domain compared with wild-type HAI-1. Overall, however, the efforts described herein helped discover and optimize the screening and structure-function analysis of HAI- 1 engineering for development of a more potent HAI-1 based matriptase inhibitor.

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