WO2023215708A1

WO2023215708A1 - Modified tissue factor with increased clotting activity

Info

Publication number: WO2023215708A1
Application number: PCT/US2023/066427
Authority: WO
Inventors: James H. Morrissey; Victor ZENG
Original assignee: The Regents Of The University Of Michigan
Priority date: 2022-05-03
Filing date: 2023-05-01
Publication date: 2023-11-09

Abstract

The present disclosure relates to modified tissue factor proteins and uses thereof. In some embodiments, the disclosure relates to modified human tissue factor proteins with increased clotting activity and uses thereof including in clotting tests, as topical hemostatic agents, and in methods of inducing tumor infarction.

Description

Atorney Docket No. UM-40837.601

MODIFIED TISSUE FACTOR WITH INCREASED CLOTTING ACTIVITY

STATEMENT REGARDING RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 63/337,691, filed May 3, 2022, the entire contents of which are incorporated herein by reference for all purposes.

STATEMENT REGARDING FEDERAL FUNDING

This invention was made with government support under HL 135823 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD

BACKGROUND

In normal hemostasis, the primary enzyme that triggers the blood clotting cascade is a membrane-bound complex of two proteins: factor Vila (FVIIa; the catalytic subunit) and tissue factor (TF; the positively-acting regulatory subunit). TF is a membrane-anchored protein expressed on the surface of many cells outside the vasculature. FVIIa is a trypsin-like serine protease that circulates in the plasma. Following vascular damage, TF binds FVIIa with high affinity. The resulting membrane-bound TF:FVIIa complex activates two serine protease zymogens via limited proteolysis: factor X (FX) and factor IX (FIX). Under most circumstances, FX is the preferred substrate over FIX.

Since TF modulates the enzymatic activity of FVIIa, TF is sometimes referred to as the protein cofactor for FVIIa. Binding of TF to FVIIa enhances the enzymatic activity of FVIIa dramatically, via allostery and other mechanisms. The “exosite” region of tissue factor (TF) facilitates macromolecular substrate binding to the TF/FVIIa complex, although the exact portion of TF which interacts with factor X (FX) remains unclear. SUMMARY

In some aspects, provided herein are modified tissue factor proteins. In some embodiments, provided herein are recombinant human tissue factor proteins. In some embodiments, the modified (e.g. recombinant) tissue factor proteins described herein possess increased clotting activity compared to wildtype tissue factor protein.

In some embodiments, provided herein is a modified human tissue factor protein comprising an amino acid sequence having 90% sequence identity with SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In some embodiments, the modified human tissue factor protein comprises one or more mutations selected from an insertion of a basic amino acid between residues 196 and 197 relative to SEQ ID NO: 2, or a substitution of the threonine at residue 197 relative to SEQ ID NO: 2 with a basic amino acid. In some embodiments, the basic amino acid comprises arginine, lysine, or histidine.

In some embodiments, the modified human tissue factor protein comprises an insertion of a basic amino acid between residues 196 and 197 relative to SEQ ID NO: 2. In some embodiments, the modified human tissue factor protein comprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

In some embodiments, the modified human tissue factor protein comprises a substitution of the threonine at residue 197 relative to SEQ ID NO: 2 with a basic amino acid. In some embodiments, the modified human tissue factor protein comprises the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

In some embodiments, the modified human tissue factor protein is conjugated to a tumor targeting moiety.

In some aspects, provided herein is a polynucleotide encoding the modified human tissue factor protein described herein. In some aspects, provided herein is a cell expressing a polynucleotide encoding the modified human tissue factor protein. In some aspects, provided herein is a system comprising a modified human tissue factor protein described herein embedded in a phospholipid bilayer. In some embodiments, the phospholipid bilayer comprises phosphatidylserine. In some embodiments, the phospholipid bilayer is part of a liposome, wherein the liposome comprises phosphatidylcholine and phosphatidylserine. In some embodiments, the liposome comprises about 80% phosphatidylcholine and about 20% phosphatidylserine.

The modified human tissue factor proteins and systems described herein find use in a variety of methods. In some embodiments, the modified human tissue factor protein or system described herein is used in a method of measuring clotting in a blood sample obtained from a subject. In some embodiments, the modified human tissue factor protein or system described herein is used in a method of treating a tumor in a subject. In some embodiments, the modified human tissue factor protein or system described herein is used in a method of controlling bleeding in a subject.

In some aspects, provided herein is a method of measuring clotting in a blood sample obtained from a subject. In some embodiments, the method comprises contacting a blood sample with a system described herein (e.g. a system comprising a modified human tissue factor protein embedded in a phospholipid bilayer), and measuring time to observable clotting in the sample.

In some embodiments, provided herein is a method of treating a tumor in a subject, comprising providing to the subject a modified human tissue factor protein or a system described herein.

In some embodiments, provided herein is a method of controlling bleeding in a subject, comprising providing to the subject a modified human tissue factor protein or a system described herein. In some embodiments, the modified human tissue factor protein or the system is administered topically to an organ or tissue within the subject.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing a comparison of initial rates of FX activation supported by human versus baboon sTF in the presence of FVIIa and PCPS vesicles. Reaction conditions were: 50 nM human or baboon sTF, 400 pM human FVIIa, 50 nM human FX, 50 μM PCPS vesicles and 0.5 mM Spectrozyme Xa (a chromogenic substrate for FXa), all in HBSA buffer (25 mM Hepes pH 7.4, 100 mM NaCl, 0.1% bovine serum albumin) plus 5 mM CaCl₂. FX was added last, to initiate the reaction. The rate of change in A₄₀₅ was measured using a Spectramax multiwell spectrophotometer (Molecular Devices) and converted to FX activation rates as described.¹⁰ Data are mean ± standard error; n=3.

FIG. 2 is a bar graph showing a comparison of initial rates of FIX activation supported by human versus baboon sTF in the presence of FVIIa and PCPS vesicles. FIX activation rates were measured using a two-stage, discontinuous assay. Briefly, 100 nM human or baboon sTF were first combined with 25 nM FVIIa and 50 μM PCPS in HBSA buffer plus 5 mM CaCl₂, after which 2 μM FIX was added to initiate the first stage. Timed, 10-μL aliquots were removed and quenched in 80 μL stop buffer (HBSA containing 20 mM EDTA and 75% ethylene glycol) on ice. After warming the quenched aliquots to room temperature, 0.5 mM Pefachrome FIXa (a FIXa chromogenic substrate) was added to initiate the second stage, and the rate of change in A₄₀₅ was quantified. FIX activation rates were calculated in reference to a standard curve using purified FIXa, as described. Data are mean ± standard error; n=2.

FIG. 3 is a graph showing a typical FVIIa titration to determine the effective memTF concentration. Reaction conditions were: 10 nM FVIIa and varying concentrations of relipidated human memTF in HBSA plus 5 mM CaCl₂, after 0.5 mM Chromozym tPA (FVIIa chromogenic substrate) was added and the rate of change in A₄₀₅ was measured. Lines were fitted separately to the data points below and above 20 nM memTF to determine the equivalence point (indicated by the vertical dotted line). The memTF concentration at this intersection point was taken to be the effective memTF concentration, which in this titration was 18 nM human memTF.

FIG. 4A-4B are graphs showing a comparison of the plasma clotting activities of relipidated human and baboon memTF. Clotting assays were conducted using a STart 4 coagulometer (Diagnostica Stago, Asnieres, France). Briefly, 50 μL of citrated pooled normal human plasma was mixed in a coagulometer cuvette with 50 μL of a solution containing varying concentrations of relipidated human or baboon memTF, then incubated for 120 sec at 37° C. Clotting was initiated by adding 50 μL of pre-warmed 25 mM CaCl₂, and the time to clot formation was recorded. FIG. 4A is a log-log plot of clotting time versus the amount of memTF in each clotting reaction (in ng). Note that the memTF amounts are plotted here on the x-axis as the effective amount of memTF calculated from FVIIa titrations as shown in Figure 3. FIG. 4B (s a bar graph showing a comparison of the specific activities of human versus baboon memTF. A unit of TF activity was defined as the amount of memTF in the clotting assay from FIG. 4A that yielded a 25-second clotting time. The calculated TF specific activities shown here are expressed as units/ng memTF protein. Data in both panels are mean ± standard error; n=3. (The error bars in FIG. 4A are small enough that most of them are covered by the data points.)

FIG. 5 shows a sequence alignment of the amino acid sequences of the TF ectodomains (sTF) from humans (SEQ ID NO: 4) and baboons (Papio anubis ) (SEQ ID NO: 21). Amino acid sequence differences are highlighted in yellow, while the single arginine insertion is highlighted in green. 93.6% identity in 220 residues overlap; Score: 1074.0; Gap frequency: 0.5%. Residue numbering is for the mature human TF ectodomain (sTF: amino acids 1-219).

FIG. 6 shows multiple sequence alignment of a portion of the TF sequence from various mammals (residues 195-202 in the human TF numbering system). Residues equivalent to Thrl97 in the human sequence bolded (and red where this is a Thr). Also, the inserted Arg residues in baboon and rhesus macaque are bolded.

FIG. 7 is a graph showing relative rates of FX activation supported by various sTF species and mutants. Using the same reaction conditions as in Figure 1, rates of FX activation supported by the indicated type of sTF in the presence of FVIIa and PCPS vesicles were quantified, and normalized to that of wild-type human sTF. The identities of the various sTF sequences are given in Table 1. Data are mean ± standard error; n=3.

FIG. 8 shows the full sequence of human tissue factor protein (SEQ ID NO: 1). The signal peptide sequence is shown underlined, the ectodomain sequence is shown in standard font, the transmembrane domain sequence is shown in bold and underlined, and the cytoplasmic domain sequence is shown in italics.

DEFINITIONS

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments described herein, some preferred methods, compositions, devices, and materials are described herein. However, before the present materials and methods are described, it is to be understood that this invention is not limited to the particular molecules, compositions, methodologies or protocols herein described, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the embodiments described herein.

Unless otherwise defined, 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. However, in case of conflict, the present specification, including definitions, will control. Accordingly, in the context of the embodiments described herein, the following definitions apply.

As used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a peptide amphiphile” is a reference to one or more peptide amphiphiles and equivalents thereof known to those skilled in the art, and so forth.

As used herein, the term “comprise” and linguistic variations thereof denote the presence of recited feature(s), element(s), method step(s), etc. without the exclusion of the presence of additional feature(s), element(s), method step(s), etc. Conversely, the term “consisting of’ and linguistic variations thereof, denotes the presence of recited feature(s), element(s), method step(s), etc. and excludes any unrecited feature(s), element(s), method step(s), etc., except for ordinarily-associated impurities. The phrase “consisting essentially of’ denotes the recited feature(s), element(s), method step(s), etc. and any additional feature(s), element(s), method step(s), etc. that do not materially affect the basic nature of the composition, system, or method. Many embodiments herein are described using open “comprising” language. Such embodiments encompass multiple closed “consisting of’ and/or “consisting essentially of’ embodiments, which may alternatively be claimed or described using such language.

As used herein, the terms “treat,” “treatment,” and “treating” refer to reducing the amount or severity of a particular condition, disease state, or symptoms thereof, in a subject presently experiencing or afflicted with the condition or disease state. The terms do not necessarily indicate complete treatment (e.g., total elimination of the condition, disease, or symptoms thereof).

As used herein, the terms “prevent,” “prevention,” and preventing” refer to reducing the likelihood of a particular condition or disease state from occurring in a subject not presently experiencing or afflicted with the condition or disease state. The terms do not necessarily indicate complete or absolute prevention. For example “preventing” refers to reducing the likelihood of a condition or disease state occurring in a subject not presently experiencing the condition or disease.

The terms “subject” and “patient” are used interchangeably herein and refer to any animal. In some embodiments, the subject is a mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha. such as rabbits, mammals from the order Carnivora, including Felines (cats) and Canines (dogs), mammals from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). In some aspects, the mam mals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). In some aspects, the mammal is a human. In some aspects, the human is an adult aged 18 years or older. In some aspects, the human is a child aged 17 years or less.

DETAILED DESCRIPTION

In some embodiments, provided herein are human tissue factor proteins. In some aspects, provided herein are modified (e.g. recombinant) human tissue factor proteins. In some embodiments, the modified human tissue factor proteins provided herein possess increased clotting activity compared to wildtype human tissue factor protein.

The full sequence of human tissue factor protein is shown in FIG. 8, with the various components thereof labeled. The full sequence is: METPAWPRVPRPETAVARTLLLGWVFAQVAGASGTTNTVAAYNLTWKSTNFKTILEW EPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVE STGS AGEPL YENSPEF TP YLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFL SLR DVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVOAVIPSRTVNRKST DSPVECMGQEKGEFREIFYIIGAVVFVVIILVIILAISLHKCRKAGVGQSWKENSPLNVS (SEQ ID NO: 1)

In some embodiments, provided herein is a modified human tissue factor protein comprising one or more amino acid modifications compared to SEQ ID NO: 1 or a portion thereof. The modified human tissue factor proteins described herein may comprise any suitable amino acid modifications, including substitutions, deletions, insertions, or combinations thereof. Amino acids 1 -32 correspond to a signal peptide sequence that is cleaved to yield what is referred to herein as the “mature” human tissue factor protein. The sequence of “mature” human TF is:

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTD TECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQ SFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTN EFLID VDKGENYCF S VQ AVIPSRTVNRKSTD SP VECMGQEKGEFREIF YIIGAVVF VVIIL VIILAISLHKCRKAGVGQSWKENSPLNVS (SEQ ID NO: 2).

In some embodiments, provided herein is a modified human tissue factor protein comprising one or more amino acid modifications compared to SEQ ID NO: 2 or a portion thereof. The modified human tissue factor proteins described herein may comprise any suitable amino acid modifications, including substitutions, deletions, insertions, or combinations thereof. In some embodiments, the modified human TF protein comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 2.

Although TF is normally a membrane-anchored protein (with a single-pass membrane anchor near its C-terminus), recombinant versions of this protein that are either a membrane- anchored (memTF) or soluble (sTF) can also be generated. In some embodiments of a recombinant memTF, the transmembrane sequence is included and at least a portion of the cytoplasmic domain, which is not required for TF clotting activity, is deleted. In some embodiments, most of the cytoplasmic domain is deleted. In some embodiments of a recombinant memTF, the transmembrane sequence is included and at least a portion of the cytoplasmic domain is included. In some embodiments, the transmembrane sequence is included and most or all of the cytoplasmic domain is included. In some embodiments, a recombinant membrane-anchored TF comprises a membrane anchoring domain (e.g. transmembrane domain) from another protein, which is substituted with the native transmembrane sequence.

An exemplary sequence for a human memTF protein is:

TTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTE CDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSF EQVGTK VNVTVEDERTLVRRNNTFLSLRD VFGKDLIYTLYYWK S S S SGKKTAKTNTNEF LIDVDKGENYCF S VQ AVIPSRTVNRKSTD SP VECMGQEKGEFREIF YIIGAVVF VVIIL VII LAISLHK (SEQ ID NO: 3)

SEQ ID NO: 3 corresponds to residues 3-244 from the mature human TF protein shown in SEQ ID NO: 2. In some embodiments, the recombinant human TF protein comprises membrane-anchored tissue factor protein comprising an amino acid sequence having at least 90% sequence identity (e.g. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) with SEQ ID NO: 3.

For recombinant sTF, all or a portion of the isolated ectodomain is expressed. The sequence of the ectodomain of human TF is as follows:

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDL TDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYIJiTNLGQPTIQSFEQV GTKWVTATDERTLVRRNNTFLSLRDWGKDLIYTLYYWKSSSSGKKTAKTNTNEFLID VDKGENYCFSVOAVIPSRWNRKSTOSPVECMGOEKGEFRE (SEQ ID NO: 4)

In some embodiments, a recombinant sTF comprises amino acids 3-219 of the mature human TF protein, shown below:

TTNTVAAYNLTWKSTNFKTTLEWEPKPVTSiQVYTVQISTKSGDWKSKCFYTTDTECDLT DEIVKDVKQTYLAR.VFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVG TK VN VTVEDERTL VRRNNTFL SLRD VFGKDLI YTLY YWK S SS SGKKT AKTNTNEFLIDV DKGENYCFSVOAVIPSRTVNRKSTDSPVECMGOEKGEFR.E (SEQ ID NO: 5)

In some embodiments, the recombinant human TF protein comprises a soluble tissue factor protein comprising an amino acid sequence having at least 90% sequence identity (e.g. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) with SEQ ID NO: 4 or SEQ ID NO: 5.

Residues underlined in bold in the above sequences (i.e. SEQ ID NO: 1 through SEQ ID NO: 5) correspond to residues 195-202 relative to the mature human TF sequence (i.e. the sequence shown in SEQ TD NO: 2). These residues comprise: SRTVNRKS (SEQ ID NO: 6). As described in Example 1, this region was identified in multiple sequence alignment as a region wherein baboon TF protein contains an arginine residue that is not present in the human sequence, which may explain the discrepancy between baboon and human TF activity. In particular, as highlighted in Example 1, baboon TF was found to have substantially higher activity than human TF, as measured both by its ability to support FX activation by FVIIa, and by its ability to trigger the clotting of human plasma. Accordingly, in some embodiments the modified proteins described herein comprise an amino acid sequence comprising one or more mutations in SEQ ID NO: 6. For example, in some embodiments the modified proteins comprise an insertion in SEQ ID NO: 6, or a substitution in SEQ ID NO: 6. In some embodiments, the modified proteins comprise a mutation in SEQ ID NO: 6 shown in Table 1.

In some embodiments, provided herein is a modified human tissue factor protein comprising an amino acid sequence having 90% sequence identity with SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In some embodiments, provided herein is a modified human tissue factor protein comprising an amino acid sequence having 90% sequence identity with SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, wherein the modified human tissue factor protein comprises an insertion of a basic amino acid between residues 196 and 197 relative to SEQ ID NO: 2. The basic amino acid may be any suitable basic amino acid, including arginine, lysine, or histidine. In some embodiments, the modified human tissue factor protein comprises an arginine residue between residues 196 and 197 relative to SEQ ID NO: 2. In some embodiments, provided herein is a modified human tissue factor protein comprising an amino acid sequence having 90% sequence identity with SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, wherein the modified human tissue factor protein comprises a substitution of the threonine at residue 197 relative to SEQ ID NO: 2 with a basic amino acid. In some embodiments, the basic amino acid is selected from arginine, lysine, and histidine. In some embodiments, the substitution comprises a T197R or a T197K substitution (positions given relative to SEQ ID NO: 2).

In some embodiments, provided herein is a modified human tissue factor protein comprising the amino acid sequence:

Such a recombinant protein is also referred to herein as the sTF insR mutant. SEQ ID NO: 7 is a soluble human tissue factor protein (sTF).

Such a recombinant protein is also referred to herein as the sTF insK mutant. SEQ ID NO: 8 is a soluble human tissue factor protein (sTF).

Such a recombinant protein is also referred to herein as the sTF insH mutant. SEQ ID NO: 9 is a soluble human tissue factor protein (sTF).

Such a recombinant protein is also referred to herein as the sTF T197R mutant SEQ ID NO: 10 is a soluble human tissue factor protein (sTF).

Such

a recombinant protein is also referred to herein as the sTF T197K mutant SEQ ID NO: 11 is a soluble human tissue factor protein (sTF).

Such a recombinant protein is also referred to herein as the sTF T197H mutant. SEQ ID NO: 12 is a soluble human tissue factor protein (sTF).

Such a recombinant protein is also referred to herein as the

memTF insR mutant. SEQ ID NO: 13 is a membrane-anchored human TF protein.

Such a recombinant protein is also referred to herein as the memTF insK mutant. SEQ ID NO: 14 is a membrane-anchored human TF protein.

Such a recombinant protein is also referred to herein as the memTF insH mutant. SEQ ID NO: 15 is a membrane-anchored human TF protein.

Such a recombinant protein is also referred to herein as the memTF T197R mutant. SEQ ID NO: 16 is a membrane-anchored human TF protein.

Such a recombinant protein is also referred to herein as the

memTF T197K mutant. SEQ ID NO: 17 is a membrane-anchored human TF protein.

Such a recombinant protein is also referred to herein as the

memTF T197H mutant. SEQ ID NO: 18 is a membrane-anchored human TF protein.

The residues shown in bold and underlined in SEQ ID NOs: 7-18 correspond to residues 195-202 relative to the mature human TF sequence (SEQ ID NO: 2) . In SEQ ID NO: 2, these residues comprise: SRTVNRKS (SEQ ID NO: 6). In the recombinant proteins show in SEQ ID NOS: 7-18, one or more mutations are made in this region of the recombinant protein (e.g. the region corresponding to residues 195-202 relative to SEQ ID NO: 2) that convey increased clotting activity to the recombinant protein

In some embodiments, a human tissue factor protein described herein is incorporated into a phospholipid bilayer. In some embodiments, provided herein is a system comprising a human tissue factor protein embedded in a phospholipid bilayer. As used herein, the term “phospholipid” refers to a molecule with a hydrophilic phosphate head and a hydrophobic lipid tail. In some embodiments, the phospholipid bilayer comprises an anionic phospholipid. Suitable anionic phospholipids include, for example, phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol (PI), and its phosphorylated derivatives the phosphoinositides (e.g. phosphatidylmositol-4-phosphate [PI4P] and phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2].) In some embodiments, the phospholipid bilayer comprises two or more different phospholipids. In some embodiments, phospholipid bilayer comprises phosphatidylserine (PS). In some embodiments, the phospholipid bilayer is part of a liposome. Accordingly, in some embodiments a human tissue factor protein described herein is incorporated into a liposome. In some embodiments, the liposome comprises phosphatidylcholine and phosphatidylserine. In some embodiments, the liposome comprises about 80% phosphatidylcholine (PC) and about 20% PS. Herein, liposomes with 80% PC, 20% PS are termed “PCPS vesicles.” Incorporation of a human tissue factor protein into a phospholipid bilayer, liposome, vesicle, etc. is also referred to herein as “relipidation”. A human tissue factor protein is referred to as “relipidated” if it is incorporated into a phospholipid bilayer, liposome, vesicle, etc.

In some embodiments, provided herein is a polynucleotide encoding a modified human tissue factor protein as described herein. In some embodiments, provided herein is a cell expressing a polynucleotide encoding a modified human tissue factor protein as described herein.

The proteins and systems described herein find use in a variety of methods. In some embodiments the proteins and systems described herein are used in methods for measuring clotting in a blood sample. For example, the proteins and systems may be used as a thromboplastin reagent in a clotting test, such as a prothrombin time (PT) clotting test. A thromboplastin or a thromboplastin reagent refers to a mixture of phospholipids and tissue factor that triggers the plasma clotting cascade, leading to the conversation of prothrombin to thrombin and the formation of a fibrin clot. The prothrombin time (PT) clotting test is widely used in clinical laboratories to measure how long it takes for a clot to form in a blood sample. A PT test finds use in a variety of methods, including to monitor the effectiveness and dosing of warfarin and related anticoagulant drugs; screen for coagulation abnormalities; assess liver function as part of the MELD score; and help in the diagnosis of disseminated intravascular coagulation (DIC). Historically, thromboplastin reagents were prepared from homogenized animal or human tissues such as brain or placenta because they contain relatively high levels of TF. Because the baseline clotting time of a typical PT test with normal plasma is around 12 seconds, it is necessary to include relatively high TF concentrations in such thromboplastin reagents. Therefore, employing a mutant human TF described herein, which possesses increased clotting activity, would reduce the amount of recombinant TF necessary per PT test.

In some embodiments, provided herein is a method comprising contacting a blood sample with a protein or system described herein. In some embodiments, the method further comprises measuring clotting in the sample. In some embodiments, measuring clotting comprises determining the amount of time that passes between contacting the blood sample with the protein or system described herein and observable clotting in the sample. In some embodiments, the modified TF proteins or systems describing the same can be used as a hemostatic agent. Accordingly, in some embodiments provided herein is a method of controlling bleeding in a subject. In some embodiments, the method comprises providing to the subject a protein or a system described herein. In some embodiments, the method comprises topically applying a protein or a system described herein to a tissue or an organ of the subject. As used herein, the term “tissue” refers to a collection cells that work together to accomplish a bodily function. For example, “tissue” is inclusive of epithelial tissue, connective tissue, nervous tissue, and muscle tissue. The term “organ” refers to any organ within the body, including brain, lungs, liver, bladder, kidneys, heart, stomach, intestines, and skin. For example, the method may comprise topically applying a protein or a system described herein to a tissue or an organ of a subject during surgery. The term “controlling bleeding” refers to reducing the amount of blood loss that would otherwise occur from the tissue or organ in the absence of the hemostatic agent. Accordingly, application of the protein or systems described herein to the tissue or the organ may reduce bleeding from the tissue or organ. For example, the method may comprise topically applying a protein or a system described herein to the liver during liver surgery, thereby controlling bleeding from the liver during said surgery. Given that the modified tissue factor proteins described herein possess increase clotting activity compared to wildtype human tissue factor protein, use of the modified proteins and systems comprising the same would provide an effective hemostatic agent even at a relatively low dose.

In some embodiments, the modified tissue factor proteins and systems comprising the same described herein may be used in methods of treating cancer. For example, the modified tissue factor proteins and systems comprising the same may be used to disrupt the flow of blood to/from a tumor, referred to herein as “tumor infarction”. Tumor infarction results in ischemia, vascular infarction, and subsequent necrosis and apoptosis of neoplastic cells, thereby treating the tumor. Accordingly, in some embodiments provided herein is a method of treating a tumor in a subject, comprising providing to the subject a modified tissue factor protein or system as described herein. The term “treating” a tumor may refer to reducing the size of a tumor or completely eliminating the tumor in the subject. In some embodiments, the modified tissue factor protein or system comprising the same is administered directly to the site of the tumor, such as by topical administration or by parenteral administration (e.g. injection). In some embodiments, the modified tissue factor protein is conjugated to a tumor targeting moiety that facilitates delivery of the modified tissue factor protein to the tumor site. For example, the tumor targeting moiety may be an antibody, an aptamer, a peptide, a nucleic acid, a carbohydrate, a small molecule, a macromolecule, a nanoparticle, or another targeting moiety. In some embodiments, the tumor targeting moiety interacts with an entity expressed on tumor cells (e.g. on the surface of the tumor cells), thereby targeting the tissue factor protein to the tumor cells. In some embodiments, the modified tissue factor protein (e.g. a tissue factor protein described herein conjugated to a targeting moiety) or the system comprising the same can be provided to the subject by any suitable means and does not necessarily need to be applied directly to the tumor site. For example, the modified tissue factor protein or system comprising the same can be administered parenterally (e.g. by injection) at a site distanced from the tumor itself.

In some aspects, provided herein are kits. In some embodiments, provided herein is a kit comprising a modified human tissue factor protein as described herein. In some embodiments, the kit further comprises additional components required for use of the kit, such as for use in a method of measuring time to observable clotting in a blood sample. For example, the kit may further comprise reagents for collection/ storage of the blood sample including syringes, vials, tubes, etc. The kit may further comprise instructions for use, which may be in printable or a web-based (e.g. online) format.

Example 1

This example demonstrates the production of recombinant human tissue factor proteins possessing increased clotting activity compared to wildtype. In particular, baboon TF was found to have substantially higher activity than human TF, as measured both by its ability to support FX activation by FVIIa, and by its ability to trigger the clotting of human plasma. Almost all of this increased procoagulant activity was determined to be due to a single amino acid difference between baboon and human TF - namely, an insertion of a single Arg residue between amino acids 196 and 197 of the human TF sequence. In examining sequence alignments of mammalian TF sequences in the vicinity of this insertion, it was also discovered that amino acid 197 in TF is a Thr in humans and some primates but is a basic residue (Arg or Lys) in most other mammals. Substituting Thrl 97 with Arg or Lys in the human sequence substantially increased TF’s procoagulant activity. Therefore, the data provided herein shows that it is possible to increase the activity of human TF almost twofold with the insertion or substitution of a single basic amino acid at or adjacent to residue 197 in the human TF sequence. Since TF has several current and potential uses in the clinic and in the clinical laboratory, enhancing the activity of human TF provides a significant improvement to its current and potential therapeutic and diagnostic uses. sTF binds with relatively high affinity to FVIIa and retains the ability to allosterically activate FVIIa. The sTF:FVIIa complex can associate with membranes because of the reversible, membrane-binding ability of FVIIa. Similarly, the two protein substrates, FX and FIX, also bind reversibly to membranes. For this reason, PCPS vesicles enhance the rate of FX or FIX activation by sTF:FVIIa. However, the affinity of FVIIa for membranes is not strong, so it typically takes higher concentrations of the sTF:FVIIa complex to activate FX, compared to the meniTF:FVIIa complex when memTF is embedded in PCPS vesicles. However, sTF is technically easier to use than memTF, so for ease of use initial experiments herein were often conducted on TF cofactor function using sTF plus PCPS vesicles.

MATERIALS

The predicted amino acid sequence of baboon (Papio arm bis) TF protein was obtained from the NCBI GenPept database, accession number XP_003892278 (version XP_003892278.1; ncbi.nlm.nih.gov/protein/XP_003892278.1). The following proteins were purchased from the indicated suppliers: recombinant human FVIIa, American Diagnostica (now Sekisui Diagnostics, Lexington, MA, USA); human plasma-derived FIX and FX, Haematologic Technologies (Essex Junction, VT, USA). Recombinant human sTF (residues 3-219) and baboon sTF (spanning the homologous residues in the baboon TF sequence), both with epitope tags, were expressed in Escherichia coli and purified.

Recombinant human memTF (residues 3-244) and baboon memTF (spanning the homologous residues in the baboon TF sequence), both with epitope tags, were expressed in E. coli and purified. The amino acid sequences of the baboon sTF and memTF expression constructs are given here: Baboon sTF amino acid sequence:

Baboon memTF amino acid sequence:

Both the sTF and memTF sequences in these E. coli expression constructs are preceded by the pelB leader peptide (underlined and in italics) which is removed during expression. The baboon sTF sequence is followed on its C-terminus with a short spacer and a 6*His epitope tag (indicated in bold) for purification purposes. The baboon memTF sequence has an HPC4 epitope tag⁸ (indicated in bold) at its N-terminus for purification purposes. The predicted transmembrane sequence of baboon memTF is indicated by bold underlining.

Phospholipids were purchased from Avanti Polar Lipids (Alabaster, AL), as follows: PC, l-palmitoyl-2-oleoyl-sw-glycero-3 -phosphocholine; PS, l-palmitoyl-2-oleoyl-sw-glycero-3- phospho-L-serine. PCPS liposomes consisted of 80% PC, 20% PS and were prepared via membrane extrusion without the addition of bovine serum albumin.

Relipidated memTF (memTF-liposomes) were prepared by incorporating memTF into phospholipid vesicles containing 80% PC and 20% PS using 15 mM deoxy cholate as the detergent. Citrated pooled normal human plasma was purchased from George King Bio-Medical (Overland Park, KS). Chromogenic substrates were purchased from the following suppliers: Spectrozyme Xa, Bachem (Bubendorf, Switzerland); Pefachrome FIXa, DSM Nutritional Products Ltd., Branch Pentapharm (Parsippany, NJ); and Chromozym tPA, Sigma- Aldrich. RESULTS

Baboon sTF supports higher rates of FX activation by FVIIa than does human sTF

Recombinant human and baboon sTF were expressed and purified, and their ability to support FX activation in the presence of FVIIa and PCPS vesicles was quantified. Baboon sTF consistently supported approximately 1.8-fold higher rates of FX activation compared with human sTF (Figure 1). Note that in this experiment, a limiting concentration of FVIIa (400 pM) and a large excess of sTF (50 nM), which is substantially above the Kd for the sTF -FVIIa binding interaction (~2 to 5 nM⁵) were used. In this way, the enzyme concentration (i.e., the concentration of the sTF:FVIIa complex) will be determined by the limiting FVIIa concentration. This eliminates uncertainty owing to the precise concentrations of baboon versus human TF, in case they are not of identical purity.

Baboon and human sTF support essentially the same rates of FIX activation

FIX is an alternate substrate for TFT Vila (although FX is the preferred substrate under most conditions.) The ability of human versus baboon sTF to support FIX activation in the presence of FVIIa and PCPS vesicles was compared. Unlike the case for FX as substrate, nearly the same rates of FIX activation supported by human versus baboon sTF (Figure 2) were found. In this experiment, limiting FVIIa and an excess of human or baboon sTF was used.

Relipidated baboon memTF has higher procoagulant activity than relipidated human 1116111 1 1

The ability of human vs. baboon memTF to clot human plasma was next compared. To do this, human and baboon memTF were relipidated in PCPS liposomes as described above. Because the efficiency of relipidation can vary, and also because the percentage of the relipidated memTF molecules that are on the outside surface of the liposomes (versus facing the lumen) can also vary, the “effective” memTF concentration in mem-TF liposomes was determined by titrating them against a fixed FVIIa concentration and quantifying the increased enzymatic activity. Figure 3 shows an example of the titration of a preparation of relipidated human memTF versus a fixed concentration of 10 nM FVIIa. In the example in Figure 3, it required 18 nM memTF to saturate 10 nM FVTIa, so the correction factor to calculate the effective memTF concentration was 10/18 = 0.556. Thus, the human memTF stock solution used in this experiment was 300 nM, but the effective memTF concentration was taken to be 167 nM and this was the concentration that was used in subsequent clotting assays. Baboon memTF preparations were titrated in the same way.

Using these titrations, the concentrations of relipidated human and baboon memTF was adjusted to reflect their effective memTF concentrations, and plasma clotting assays were then conducted to compare their clotting activities. As shown in Figure 4A, relipidated baboon memTF in PCPS vesicles consistently exhibited shorter clotting times than did human memTF across a range of TF concentrations. Using a unit definition for TF clotting activity as the amount of TF that yielded a 25-second clotting time, the specific activity of the two memTF preparations was calculated. Relipidated baboon memTF had an approximately twofold higher specific activity compared to human memTF (Figure 4B).

An alignment of the amino acid sequences of the human and baboon TF ectodomains revealed 93.6% sequence identity, with 13 amino acid substitutions between human versus baboon, plus one amino acid insertion — an Arg residue between amino acids 196 and 197 of the human TF sequence (Figure 5). From the published x-ray crystal structures of the sTF:FVIIa complex (Banner DW, D'Arcy A, Chene C, et al. The crystal structure of the complex of blood coagulation factor Vila with soluble tissue factor. Nature. 1996; 380: 41-46), the amino acid substitutions between human and baboon TF occurred in regions that seemed unlikely to be directly involved in allosteric activation of F Vila or in interaction with FX or FIX. On the other hand, the Arg residue inserted between residues 196 and 197 is located on the same face of TF as the substrate-binding “exosite” region of TF, which functions to facilitate macromolecular substrate binding to the TF :FVIIa complex. Accordingly, this Arg insertion might explain the increased activity of baboon tissue factor. If so, it would suggest that the TF exosite in baboon TF is more extensive than the exosite region previously mapped in human TF using alanine- scanning mutagenesis.

A multiple sequence alignment of a portion of the TF sequence among several mammalian species (Figure 6, encompassing residues 195-202 of the human TF sequence) revealed that residue 197 is a Thr in some primates but is a basic residue (Arg or Lys) in almost all other mammals. Furthermore, while baboons and rhesus macaques have a Thr at this position, they also have an inserted Arg residue just before it, relative to the human sequence. Great apes and humans, on the other hand, merely have the Thr residue at position 197, without the Arg insertion. Accordingly, changing residue Thr 197 in human TF to Arg or Lys may increase its procoagulant activity in a manner similar to that of inserting an Arg between residues 196 and 197.

Inserting an Arg between residues 196 and 197 of human sTF increases the rate of FX activation by the sTF:FVIIa complex, as does changing Thrl97 to a basic residue (Lys or Arg).

Starting with the human sTF sequence, a series of mutants centered on the Thrl97 residue (Table 1).

*Differences from the human sTF sequence are indicated in bold. FX activation rates for the sTF constructs in Table 1 were measured in the presence of FVIIa and PCPS vesicles (Figure 7). Baboon sTF supported an approximately 1.7-fold higher rate of FX activation than did human sTF. Inserting an Arg between residues 196 and 197 (insR) resulted in an approximately 1.7-fold higher rate of FX activation compared to wild-type sTF. Replacing Thrl97 with Arg (T197R) or Lys (T197K) resulted in an approximately 1.5-fold and 1.6-fold increases, respectively, compared to wild-type sTF. On the other hand, replacing Thrl97 with Ala (T197A) resulted in approximately the same rate of FX activation as seen with wildtype sTF.

Claims

CLAIMS We claim:

1. A modified human tissue factor protein comprising an amino acid sequence having 90% sequence identity with SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, wherein the modified human tissue factor protein comprises one or more mutations selected from an insertion of a basic amino acid between residues 196 and 197 relative to SEQ ID NO: 2, and a substitution of the threonine at residue 197 relative to SEQ ID NO: 2 with a basic amino acid.

2. The modified human tissue factor protein of claim I, wherein the basic amino acid comprises arginine, lysine, or histidine.

3. The modified human tissue factor protein of claim 1, wherein the modified human tissue factor protein comprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

4. The modified human tissue factor protein of claim 1, wherein the modified human tissue factor protein comprises the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

5. The modified human tissue factor protein of any one of claims 1-4, wherein the modified human tissue factor protein is conjugated to a tumor targeting moiety.

6. A polynucleotide encoding the modified human tissue factor protein of any one of claims 1-5.

7. A cell expressing the polynucleotide of claim 6.

8. A system comprising the modified human tissue factor protein of any one of claims 1 - 5 embedded in a phospholipid bilayer.

9. The system of claim 8, wherein the phospholipid bilayer comprises phosphati dyl serine .

10. The system of claim 8 or claim 9, wherein the phospholipid bilayer is part of a liposome, wherein the liposome comprises phosphatidylcholine and phosphatidylserine .

11. The system of claim 10, wherein the liposome comprises about 80% phosphatidylcholine and about 20% phosphatidyl serine.

12. The modified human tissue factor protein of any one of claims 1-5 or the system of any one of claims 8-11, for use in a method of measuring clotting in a blood sample obtained from a subject.

13. The modified human tissue factor protein of any one of claims 1-5 or the system of any one of claims 8-11, for use in a method of treating a tumor in a subject.

14. The modified human tissue factor protein of any one of claims 1-5 or the system of any one of claims 8-11, for use in a method of controlling bleeding in a subject.

15. A method comprising contacting a blood sample with the system of any one of claims 8-11, and measuring time to observable clotting in the sample.

16. A method of treating a tumor in a subject, comprising providing to the subject the modified human tissue factor protein of any one of claims 1-5, or the system of any one of claims 8-11.

17. A method of controlling bleeding in a subject, comprising providing to the subject the modified human tissue factor protein of any one of claims 1-5, or the system of any one of claims 8-11.

18. The method of claim 17, wherein the modified human tissue factor protein or the system is administered topically to an organ or tissue within the subject.

19. Use of the modified issue factor protein of any one of claims 1-5 in a method of measuring clotting in a blood sample obtained from a subject.

20. Use of the modified issue factor protein of any one of claims 1-5 in a method of treating a tumor in a subject.

21. Use of the modified issue factor protein of any one of claims 1-5 in a method of controlling bleeding in a subject.

22. Use of the system of any one of claims 8-11 in a method of measuring clotting in a blood sample obtained from a subject.

23. Use of the system of any one of claims 8-11 in a method of treating a tumor in a subject.

24. Use of the system of any one of claims 8-11 in a method of controlling bleeding in a subject.