WO2008005071A1

WO2008005071A1 - Cell-based assay for measuring anti-proliferative activity of oxidized tfpi

Info

Publication number: WO2008005071A1
Application number: PCT/US2007/005851
Authority: WO
Inventors: Glenn Deaver; David Fei
Original assignee: Novartis Ag
Priority date: 2006-03-03
Filing date: 2007-03-05
Publication date: 2008-01-10

Abstract

A cell-based assay for measuring anti-proliferative activity of oxidized TFPI.

Description

CELL-BASED ASSAY FOR MEASURING ANTI-PROLIFERATIVE ACTIVITY OF OXIDIZED TFPI

FIELD OF THE INVENTION

[01] The invention relates to methods of determining TFPI oxidation.

BACKGROUND

[02] Tissue Factor Pathway Inhibitor (TFPI), which contains three Kunitz-type proteinase inhibitor domains (FIG. 1), is well known to be an important inhibitor of blood coagulation (reviewed in Price et al., Anesthesia 59, 483-02, 2004). Kunitz domain 1 inhibits the formation of fibrin clots via the extrinsic pathway by binding to the tissue factor/factor Vila complex. Kunitz domain 2 binds factor Xa and directly inhibits fibrin clot formation via the intrinsic pathway.

[03] Use of TFPI has been proposed for the treatment of various indications, including sepsis (U.S. 6,063,764 and WO 93/24143), deep vein thrombosis (U.S. 5,563,123, U.S. 5,589,359, and WO 96/04378), ischemia (U.S. 5,885,781, U.S. 6,242,414, and WO 96/40224), restenosis (U.S. 5,824,644 and WO 96/01649), and cancer (U.S. 5,902,582 and WO 97/09063). A TFPI variant, which differs from TFPI by the addition of an alanine residue at the amino terminus ("ala-TFPI"), is efficacious in animal models for the treatment of sepsis. Carr et al., Circ Shock 1994 Nov;44(3): 126-37.

[04] There are five methionine residues in a TFPI molecule, located at positions 23, 40, 76, 135, and 277 of SEQ ID NO:1. The methionine residues are the sites at which oxidation occurs. Oxidation is known to reduce biological activity in some recombinant proteins (see for example, Griffiths (2002) Journal of Biological Chemistry 277(28): 25486). There is a need in the art for methods of determining TFPI oxidation, for example, to help ensure that manufacturing lots of therapeutic preparations of TFPI have optimal biological activity. SUMMARY OF THE INVENTION

[05] The invention provides a quantitative or qualitative cell-based bioassay which can be used to determine the oxidation level of TFPI and which can be used, e.g., as biological activity-indicating lot release assay for quality control of recombinant TFPI (rTFPI). TFPI induces apoptosis in cultured endothelial cells, and thus the methods utilize an assay system that monitors cell proliferation. The bioassay is particularly suited to monitoring the extent of oxidation present in a lot of rTFPI since increased TFPI oxidation leads to a decrease in TFPI' s anti-proliferative activity.

[06] Accordingly, in one embodiment, the invention is directed to an in vitro method for determining TFPI oxidation. The method comprises:

(a) determining a first measure of cell proliferation in a first population of proliferating endothelial cells in vitro in the presence of the TFPI;

(b) comparing the first measure with a second measure of cell proliferation in a second population of the proliferating endothelial cells in the absence of TFPI to determine a first percent inhibition of cell proliferation; and

(c) identifying the TFPI as oxidized if the first percent inhibition of cell proliferation is lower than a second percent inhibition of cell proliferation determined using TFPI which is not oxidized.

[07] In certain embodiments of the method, step (a) is carried out in the presence of 1.0 μM TFPI.

[08] In additional embodiments of the method, the TFPI is ala-TFPI.

[09] In still further embodiments, the endothelial cells used in the method are human umbilical vein endothelial cells (HUVEC).

[10] In other embodiments, proliferation of the endothelial cells is stimulated using fibroblast growth factor 2 (FGF-2).

[11] In yet an additional embodiment, proliferation is determined by detecting [12] In certain embodiments of the subject invention, the method is determined quantitatively.

[13] These and other embodiments of the subject invention will readily occur to those of skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[14] FIG. 1. Illustration showing amino acid sequence (SEQ ID NO:1) and Kunitz domains of TFPI.

[15] FIG. 2. Graph showing oxidized rTFPI inhibition of proliferation of HUVEC.

[16] FIG. 3. Graph showing inhibition of HUVEC proliferation at 1 μM rTFPI.

DETAILED DESCRIPTION OF THE INVENTION

[17] The invention provides a cell-based bioassay which can be used to determine the oxidation level of TFPI and which can be used, e.g., as biological activity-indicating lot release assay for quality control of recombinant TFPI (rTFPI).

[18] TFPI induces apoptosis in cultured endothelial cells (see for example Hamuro et al., FEBS Lett. 421, 197-202, 1997; Hembrough et al, J. Biol. Chem. 267, 12241-48, 2001). Assays of the invention take advantage of TFPI' s effect on apoptosis. Proliferating endothelial cells are cultured in the presence of TFPI. The amount of cell proliferation in the cultures is then determined and compared with the amount of cell proliferation in a population of proliferating endothelial cells cultured in the absence of TFPI. We have determined that the extent of oxidation present in a lot of TFPI has a dose-dependent affect on the inhibition of endothelial cell proliferation that occurs as a result of exposure to TPFI, i.e., more oxidized TFPI has less antiproliferative activity than TFPI that has a smaller amount of oxidation. Thus, oxidized TFPI can be detected if the percent inhibition of cell proliferation in the experimental sample is lower than the percent inhibition of cell proliferation determined using control TFPI which is not oxidized. Such assays can be used qualitatively or quantitatively, to determine the degree of oxidation of TFPI.

[19] "TFPI" as used herein includes TFPI as shown in SEQ ID NO:1 and ala-TFPI (TFPI example as described in U.S. 4,966,852 or US 2005/0037475. Alternatively, TFPI can be purified from plasma, for example as described in Novotny et al., Blood 1991 JuI 15;78(2):394-400.

[20] Cell proliferation can be determined by any means known in the art, including, but not limited to, measuring increases in plasma membrane permeability, reduction in mitochondrial metabolic activity, fragmentation of DNA (e.g., by size or detection of histone-complexed DNA fragments), alterations in membrane asymmetry, activation of apoptotic caspases (e.g., by in vitro enzyme assay), mitochondrial release of AIF or cytochrome C into the cytoplasm, incorporation of radiolabeled thymidine, and staining with various dyes, such as ALAMARBLUE™, tetrazolium salts 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and (2,3-bis(2- methoxy-4-nitro-5- sulfophenyl)-2H-tetrazolium-5-carboxanilide) (XTT), and neutral red.

[21] One embodiment of the assay is described in the Examples below. This embodiment uses human vascular endothelial cells (HUVEC); however, any type of endothelial cell which proliferates in vitro can be used. Vascular endothelial cells are particularly useful. Vascular endothelial cells can be derived from any part of the vascular tree, including large and small veins and arteries, capillaries, the umbilical vein of newborns (e.g., HUVEC), cerebral blood vessels, or even from vascularized solid tumors.

[22] All patents, patent applications, and references cited in this disclosure are expressly incorporated herein by reference. The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples, which are provided for purposes of illustration only and are not intended to limit the scope of the invention. EXAMPLE l

[23] Materials. HUVEC cells, Medium 200PRF, and low serum growth supplement (LSGS) were obtained from Cascade Biologies (Portland, OR). Fetal bovine serum (FBS) was obtained from JRH Biosciences (Lenexa, KS). Penicillin-streptomycin was obtained from Mediatech, Inc. (Herndon, VA). The SpectraMax Gemini EM fluorometer was obtained from Molecular Devices Corporation (Sunnyvale, CA). Microtiter plates were manufactured by Corning, Inc. (Corning, NY). TFPI and FGF- 2 were expressed recombinantly. ALAMARBLUE™ assay reagent was purchased from Biosource International (Camarillo, CA). "Plating medium" was Medium 200PRF containing 2% FBS and 1% penicillin-streptomycin. "Assay medium" was plating medium with the addition of 10 ng/ml rFGF-2. The FGF concentration can be increased to over 100 μg/ml.

[24] TFPI Samples. Recombinant ala-TFPI ("rTFPI") tested for antiproliferative activity included 3%, 8%, 22%, and 100% oxidized TFPI. The 3% oxidized rTFPI was freshly manufactured material. The 8% and 22% oxidized rTFPI were representative samples stored at 2-8⁰C for varying time points over 18 months. The oxidation levels of the 3%, 8%, and 22% oxidized rTFPI were determined by reverse phase high performance liquid chromatography (RP-HPLC).

[25] Fully (100%) oxidized rTFPI was prepared by reacting rTFPI with 0.1% hydrogen peroxide at room temperature for 24 hours. The rTFPI was then purified using a tangential flow system to remove the residual hydrogen peroxide. The hydrogen peroxide-free rTFPI was then concentrated to 20.7 mg/ml. The fully oxidized rTFPI was characterized by RP-HPLC and LC-MS. An 80 Dalton increase in mass confirmed that all five methionines had been oxidized to methionine sulfoxide.

[26] Culture of HUVEC. HUVEC cultures were grown as specified by Cascade Biologies. Briefly, frozen cells were seeded at 2500-5000 cells/cm² in T75 Costar tissue culture flasks containing Medium 200PRF with LSGS and antibiotics and incubated at 37⁰C, 5% CO₂. Growth medium was changed following an overnight incubation and every 2 days thereafter until the cultures reached 80% confluence. Medium was then changed every day until cells were either passaged as described above or used for testing rTFPI antiproliferative activity. [27] Preparation of TFPI for testing. TFPI samples are diluted to 2 μM in assay medium and added to a separate dilution plate. The TFPI is then serially diluted either 1 :2 or 1:67 down to 250- 500 nM.

[28] Staining conditions. The viable stain (10% ALAMARBLUE™ in plating medium) is prepared and warmed to 37 ⁰C. Culture medium is removed from assay plates and replaced with the staining solution. Alternatively, ALAMARBLUE™ is added directly to the diluted TPFI samples at a final concentration of 10%. Following an additional 4- 8-hour incubation assay, plates were read on a fluorometer using excitation and emission wavelengths of 530 nm and 590 ran, respectively.

EXAMPLE 2

[29] HUVEC cells were seeded at 5,000 cells/well in 96- well assay plates in plating medium. Assay plates were incubated for 2-24 hours at 37 ⁰C, 5% CO2-

[30] Following equilibration at 37°C, 5% CO₂, plating medium was removed from assay plates. rTFPI containing assay medium was transferred to the assay plates from the dilution plates. The assay plates were then returned to the incubator for 48 hours.

[31] Each of the sample rTFPI materials was measured in triplicate in each of six assays. Percent inhibition of cell proliferation was calculated by determining:

(fluorescence of FGF-2-treated cells> - (fluorescence of FGF-2 and rTFPI-treated cells) x 100

(fluorescence of FGF-2-treated cells)

Fluorescence measurements at each rTFPI concentration of each material were averaged to obtain a single value for each assay.

[32] Mean fluorescence readings are shown in Table 1. A graph of mean fluorescence readings from a single assay is shown in FIG. 2. Mean % inhibition of cell proliferation is shown in Table 2. Summaries of mean % inhibition at 0.5 μM, 1.0 μM, and 2.0 μM rTFPI are shown in Tables 3A, 4A₅ and 5A, respectively. Means of paired samples at these same rTFPI concentrations were analyzed using two-tailed t- tests. The resulting P-values are shown in Tables 3B, 4B, and 5B. The % inhibition at 1.0 μM rTFPI is summarized in the bar graph shown in FIG. 3. Standard deviation of measured inhibition between assays is less than 6% for all oxidized rTFPI samples. [33] The results described above demonstrate that the antiproliferative activity of TFPI decreases with oxidation. In fact, when carried out in the presence of about 1 μM TFPI, different degrees of oxidation of TFPI can be distinguished. At lower TFPI concentrations, where the anti-proliferative effect of TFPI is small, there is little discernable difference between samples tested (Table 3B). Significant differences in sample activity occur at higher TFPI concentrations, with the largest difference observed at 1.0 μM TFPI (Table 4B). Differences in anti-proliferative activity at 2.0 μM TFPI were less pronounced at lower oxidation levels.

[34] While not wishing to be bound by this explanation, it is possible that saturation of the very low-density lipoprotein receptor (VLDL receptor) by non-oxidized C-terminal TFPI is responsible for this less pronounced difference. TFPI' s anti-proliferative activity reportedly is mediated by binding to VLDL receptors expressed on HUVEC. Truncated rTFPI (1-160), which contains only Kunitz domains 1 and 2, has reduced anti-proliferative activity compared to full-length rTFPI, which indicates that binding to the VLDL receptor takes place via the C-terminal portion of the molecule (Hembrough et al., 2001).

[35] The 100% oxidized sample at 1 μM concentration was least effective in inhibiting cell proliferation. While not wishing to be bound by this explanation, this effect may be because the C-terminal methionine was completely oxidized in this sample. At lower oxidation levels it is likely that oxidation was distributed heterogeneously throughout the five methionine residues resulting in increased anti-proliferative activity in those samples.

EXAMPLE 3

[37] This example confirms that the assay disclosed herein measures loss of biological function associated with TFPI oxidation.

[38] A panel of TFPI samples degraded by various means was tested in the HUVEC assay described in Example 1.

Data was processed using a 4 parameter logistic curve fitting algorithm that measures the offset of the ED50 values to determine % activity of test samples. Percent activity is a measure of the antiproliferative effect of TFPI. Percent activity of test samples was measured relative to a standard included on each assay plate.

[39] The test samples included: (1) TFPI held for 14 days at pH 2.5 or 8.5; (2) TFPI held at 40⁰C for 14 or 30 days; (3) TFPI exposed to 1.2 x 10^Λ6 lux hours; (4) TFPI mechanically agitated for 7 days on a shaker; and (5) TFPI degraded by chloramine-T treatment at 1 :1 and 10:1 molar ratios (i.e., oxidized TFPI). Data was processed using a four parameter logistic curve fitting algorithm that measures the offset of the ED50 values to determine percent activity of test samples. Percent activity is a measure of the anti-proliferative effect of TFPI. Percent activity of test samples was measured relative to a standard included on each assay plate.

[40] Of these test samples, the HUVEC assay was able to measure loss of anti-proliferative activity in the light-degraded and chloramine-T degraded samples. Results from these assays are shown in Table 6, in which the anti-proliferative effect of the test samples is expressed as % activity of fresh TFPI containing only background levels of oxidation. The samples were characterized by RP-HPLC assay coupled with Q-Tof MS and found to contain high levels of oxidized species. Table 6.

%

Sample Assay 1 2 3 Mean RSD Activity

Light 1 130 151 125 135 10 70

Degradation 2 138 164 146 149 9 78

3 129 153 136 139 9 73

4 127 137 143 136 6 71

5 132 123 130 128 4 67

6 116 125 132 124 6 65

7 133 138 145 139 4 72

8 144 146 131 140 6 73

Mean 71

RSD 6 ug/ml

Replicate #

%

Sample Assay 1 2 3 Mean RSD Activity

Chloramine-

T 1 147 174 165 162 8 84

1 :1 2 167 152 161 160 5 83

3 160 153 171 161 6 84

4 163 168 180 170 5 89

5 169 175 172 2 90

6 155 166 187 169 10 88

7 156 174 174 168 6 88

8 178 176 160 171 6 89

Mean 87

RSD 3 ug/ml

Replicate #

%

Sample Assay 1 2 3 Mean RSD Activity

Chloramine-

T 1 100 114 137 117 16 61

10:1 2 123 136 130 7 67

3 135 122 123 127 6 66

4 112 115 101 109 7 57

5 144 109 127 20 66

6 135 105 120 18 63

7 123 124 107 118 8 61

8 140 112 104 119 16 62

Mean 63

RSD 5

Claims

THAT WHICH IS CLAMED

1. A method of determining oxidation of Tissue Factor Pathway Inhibitor (TFPI), comprising:

2. The method of embodiment 1 wherein step (a) is carried out in the presence of about 1.0 μM TFPI.

3. The method of embodiment 1 wherein the TFPI is ala-TFPI.

4. The method of embodiment 1 wherein the endothelial cells are human umbilical vein endothelial cells (HUVEC).

5. The method of embodiment 1 wherein proliferation of the endothelial cells is stimulated using fibroblast growth factor 2 (FGF-2).

6. The method of embodiment 1 wherein proliferation is determined by detecting ALAMARBLUE™ fluorescence.

7. The method of embodiment 1 which is determined quantitatively.