AU657096B2 - Receptor assay for FK-506 - Google Patents

Description

RECEPTOR ASSAY FOR FK-506 BACKGROUND OF THE INVENTION

Field of the Invention

The invention relates to assays for drugs in biological fluids, more particularly to a competitive receptor protein binding assay for the

immunosuppressive drug, FK-506, and its biologically- active metabolites, derivatives and analogues

Description of the Background Art

Cyclosporine, a cyclic undecapeptide

immunosuppressive drug of fungal origin, is in

widespread use as an anti-rejection drug for solid organ transplant patients. Cyclosporine (CsA) appears to be T-cell specific and induces its immunosuppressive effects by impairing interleukin-2 production and receptor expression.¹ In the dosages required to prevent organ rejection in patients, CsA is known to exhibit serious toxic side effects, inducing

gastrointestinal and metabolic problems,

hypercholesterolemia, neurological signs and symptoms, and nephrotoxicity.^2-6 As a result, there has been an active search for substitute immunosuppressive drugs that are more efficacious at lower dosages than CsA and that have fewer toxic side effects. A compound known as FK-506 is a such a drug. FK-506 is a 822 kDa macrolide antibiotic isolated from the fungus Streptomyces tsukubaensis by the

Fujisawa Pharmaceutical Co. of Japan.⁷ Although totally different than CsA in structure (see structure I).

FK-506 exhibits similar, if not identical, antilymphocytic activities. The immunosuppressive activity of FK-506 is, however, about 100-fold greater than that of CsA,⁸ and it is being used successfully in solid organ transplant patients, particularly in liver transplant patients.⁹ Although the toxicity of FK-506 in human patients and experimental animals has been reported to be less than that of CsA⁸'¹⁰ there are reports of undesirable side effects of FK-506 in those species at the dosages required for anti-rejection activity.^2-4,10

As a result of the need to balance anti-rejection dosage requirements with the avoidance of serious, toxic side effects, dosages and plasma concentrations of FK-506, just as of CsA, must be carefully and constantly monitored.¹¹ This requires monitoring the plasma concentration, not only of the parent FK-506, but also of biologically-active metabolites of FK-506 produced, it is reported, by hepatic metabolism.¹² Thus, there is an important need for a precise, sensitive, specific, and rapid and inexpensive assay method for FK-506 and its biologically-active

metabolites, as well as of biologically-active

analogues and derivatives to be used for

pharmacokinetic studies and for setting doses for administration to patients.

Tamura et al.¹³ and Cadoff et al.¹⁴ have disclosed solid state, one-step and two-step enzyme-linked immunoassays (ELISA) for FK-506 that employ rabbit polyclonal or mouse monoclonal antibodies raised against a bovine serum albumin-FK-506 complex antigen. Several problems with this method have been reported: (1) its sensitivity is heavily dependent upon the plasma concentration of FK-506, requiring prior extraction of the drug from patient plasma by organic solvents such as benzene; (2) an inability of

distinguishing between FK-506 and certain of its metabolites that may be immunologically, but not biologically, active; (3) low precision because of the low concentration of the drug in plasma and because of the inexactness of an ELISA^;14 and (4) a lack of

correlation between dosage and plasma concentration determined by this assay.¹⁴

Zeevi et al.¹⁵ have developed a bioassay for plasma FK-506 based upon the inhibition by this drug of an alloreactive T-cell clone proliferative response.¹⁵ These authors reported that, for all plasma samples tested, the values obtained for FK-506 by the

aforementioned ELISA method,¹³'¹⁴ were consistently much higher than obtained by the bioassay. This suggests that patient blood may contain biologically inactive metabolites of FK-506 that are detected by an ELISA, but not by a bioassay. That is, as the ELISA is an immunoassay that will detect any molecule with the appropriate epitopic sites, even biologically-inactive metabolites may cross-react with an anti-FK-506 antibody.

There are several problems in such a bioassay for FK-506. This assay does not distinguish among

immunosuppressive drugs. Thus, steroids such as prednisone that are given concurrently with FK-506 (or CsA) to prevent allograft rejections, are

immunosuppressive and will react positively in any bioassay based upon the suppression of T-cell

proliferation. In addition, bioassays are not readily adaptable to routine use in hospital clinical

laboratories: the through-put of data is low in a bioassay; bioassays are slow and labor-intensive; and, biological variations among cells in tissue culture make such cells less-than-desireable as a reagent.

Thus, an important need still exists for a rapid, inexpensive, precise, sensitive and specific assay for FK-506 and its biologically-active metabolites, derivatives and analogues. Such an assay has now been discovered and is disclosed below.

SUMMARY OF THE INVENTION

A new method for the quantification of FK-506 and FK-506-like functional activity in biological fluid samples has been discovered. This method comprises a competitive protein binding assay wherein the binding reagent is a purified soluble receptor protein isolated from a target tissue of FK-506 action.

It is thus an object of this invention to provide a purified water-soluble receptor protein specific for FK-506 and its biologically-active metabolites, derivatives and analogues for use in a competitive binding assay.

It is a further object to provide aqueous

solution-based and solid state-based competitive receptor binding assays for FK-506 and its

biologically-active metabolites, derivatives and analogues using the aforementioned purified receptor protein.

It is yet another object of the invention to provide labeled FK-506 molecules suitable for use in the competitive receptor binding assay of the

invention.

These and other objects of the invention will become apparent from the following description of the preferred embodiments thereof and from the claims.

DETAILED DESCRIPTION OF THE DRAWINGS

FIGURE 1 compares the binding of FK-506 and CsA to CsA binding proteins in CEM lymphocyte cytosol. FIGURE 2 shows specific binding of FK-506 to a CEM lymphocyte cytosolic protein.

FIGURE 3 shows the elution profile of FK-506-binding protein fractions fractionated from CEM lymphocyte cytosol by HPLC molecular weight sieving chromatography on a BioRad TSK column.

FIGURE 4 shows the fractionation by Beckman HPLC on a

BioRad BioSil SEC 125 column of FK-506 and CsA binding proteins derived from JURKAT

lymphocyte cytosol. Molecular weight markers are also shown.

FIGURE 5 shows the elution profile of JURKAT lymphocyte FK-506 and CsA binding proteins from a

Matrex Gel Blue A affinity column as a function of salt concentration.

FIGURES 6A-B show the HPLC fractionation on a BioRad SEC125 molecular weight exclusion column of pooled CsA and FK-506 binding fractions isolated by Matrex Blue A affinity chromatography. Fig . 6A shows the size exclusion profile of cyclophilin (about 17 kDa), and 6B that for the about 8-12 kDa FK-506 receptor protein.

FIGURE 7 shows the fractionation of 17 kDa cyclophilin from Fig. 6A on a weak cation exchange chromatography HPLC column (Beckman TSK CM-25W Spherogel). FIGURE 8 shows the fractionation of the FK-506 8-10 kDa binding protein from Fig. 6B on a weak cation exchange chromatography HPLC column (Beckman TSK CM-25W

Spherogel). FIGURE 9 shows the fractionation of about 50 kDa proteins eluted from a size exclusion column on a weak cation exchange column.

FIGURE 10 shows analytical SDS-PAGE of the purified JURKAT about 8-12 kDa receptor protein.

FIGURE 11A-C show Scatchard plots for CsA binding for the about 50 kDa protein preparation (Fig. 11A), FK-506 binding to the same preparation (Fig. 11B), and FK-506 binding to its about 8-12 kDa receptor protein (Fig. 11C).

FIGURE 12A-B show standard curves for the receptor assay of FK-506 in water (Fig. 12A) and in whole human blood (Fig. 12B) using the purified 8-12 kDa JURKAT cell receptor protein as binding reagent. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

There is general agreement that the term

"receptor" refers to a macromolecular protein or glycoprotein capable of recognizing and selectively binding with some ligand, and which, after binding the ligand, is capable of generating some chemical or physical signal that initiates the chain of events leading to the biological response. Blecher, M., et al., "Receptors and Human Disease," Williams & Wilkens, Baltimore, 1981, Chapt. 1.

It is thus an important aspect of this invention that the water-soluble binding protein used as a reagent in the competitive receptor binding assay

(CRBA) for FK-506 and its biologically-active

metabolites, analogues and derivatives (hereinafter collectively referred to as FK-506) has several of the characteristics of a natural receptor protein, i.e., specific, saturable and reversible binding.

Water-soluble, specific receptor proteins suitable for CRBAs carried out in accordance with this invention may be advantageously prepared from extracts of target tissues of FK-506 action, such as mammalian

lymphocytes, including human or animal peripheral blood lymphocytes (PBL),¹⁶ primary mixed lymphocytes cultures, proliferating alloreactive T-cells propagated from organ transplant biopsies,¹⁵'¹⁷ and established cell lines such as the DQwl-specific alloreactive T-cell clone DB29,¹⁸ a transformed T-helper cell line (CEM) and the interleukin-2-producing JURKAT human lymphocytic leukemia T-cell line (JURKAT 77.6.8).¹⁹ As will be shown below, and has been reported elsewhere, ¹⁹ FK-506 and CsA do not share binding proteins in common. In addition, FK-506 does not bind to, and inhibit the peptidyl-prolyl cis-trans isomerase of the 17 kDa cyclophilin fraction, although it does do so to the isomerase activity of the JURKAT T-cell about 8-12 kDa FK-506 binding protein.¹⁹. This enzyme, which is highly sensitive to CsA is thought to be a primary receptor for CsA action.^20-22

It should be emphasized that the particular cellular source of the receptor protein used in the CRBA of the invention is not critical - the protein need only exhibit the binding properties of a

physiological receptor detailed above.

Cells may be disrupted and the soluble proteins (cytosolic proteins) fractionated by art-recognized methods. Typically, lymphocytes are collected, washed and counted. Cells may be stored as a pellet at -70°C until used. Cells are thawed and homogenized with a Teflon or ground glass homogenizer at ice bath

temperatures. Thoroughness of cell disruption can be tested by the trypan blue exclusion method.

Homogenates are centrifuged in the cold under reduced pressure at least 20,000 x g, preferably at least

100,000 x g, for at least 0.5 hrs. The supernatant fluid, which contains both the FK-506 receptor protein and cyclophilin is analyzed for protein content and binding activity and is either used immediately or stored frozen at temperatures at or below about -20°C.

Cytosols can be used as such for the CRBA carried out in accordance with the invention. Preferably, however, FK-506 receptor protein is purified from the cytosol and concentrated prior to use. FK-506 receptor protein can be partially purified by affinity

chromatography. A preferred affinity column is Matrix Gel Blue A (Amicon Corp., Danvers, MA) on which FK-506 and CsA binding proteins are readily separable by salt buffer gradients. Cytosol can also be fractionated by molecular weight exclusion methods, including HPLC columns. For example, a preferred molecular sieve method uses a Beckman Instrument Co. HPLC instrument and a preparative Biorad Biosil SEC 125 column of appropriate dimension (see, Example 6, below). FK-506 receptor protein can also be advantageously purified by cation exchange chromatography, e.g., by the weak cation exchange Beckman TSK CM-25W Spherogel.

Hydrophobic interaction chromatographic matrices are also suitable for purifying the FK-506 according to this invention. Any sequences or combinations of these fractionation systems can be used according to this invention as long as appropriate purifications are obtained. For example, purification by the sequence: cytosol, affinity matrix, molecular weight exclusion gel and weak cation exchanger is particularly

preferred. For the purposes of the assay of this invention, a receptor protein is deemed to be purified if, upon chromatography, a single peak describes the elution pattern of both protein and binding activity, and if, upon SDS-PAGE, only one major protein band appears. Fractions of column eluate are collected, pooled, and concentrated in the cold by rotary

evaporation at reduced pressure. Concentrates are assayed for protein by any suitable method, including the Bradford BCA method. Concentrates are also tested for assay suitability by the CRBA.

It should be emphasized that a FK-506 receptor protein may be prepared and purified by techniques other than by isolation from mammalian target cells.

For example, the receptor protein may advantageously be synthesized by art-recognized recombinant DNA

techniques.²³ In brief, cDNA coding for the receptor may be isolated from purified mRNA or from a cDNA cloning library, then cloned into cells, e.g.,

prokaryotic cells, to produce large amounts of the cDNA. An expression vector may be constructed

containing the cDNA for a FK-506 receptor, promoter and gene regulation sequences, a translation start codon, selectable markers, etc., and then inserted into prokaryotic or transformed eukaryotic cells. These cells will express the receptor gene and secrete the receptor protein in large quantities into the growth medium, from which it can be isolated by conventional methods.

For the purpose of a CRBA according to this invention, a protein fraction is deemed acceptable if: (1) the protein binds FK-506 to a statistically

significant extent based upon the method of detection. e.g., radioactivity, fluorescence polarization, chemiluminescence and the like; (2) unlabeled and labeled FK-506 compete with each other for specific binding sites on the receptor protein; and (3) the signal-to-noise ratio, i.e., the ratio of total binding to nonspecific binding (as these terms are defined below) is at least 1.1, preferably at least about 1.2.

Gel electrophoresis and Western blot analyses may be used to monitor the purity of, and identify the receptor protein throughout the stages of purification.

Labeled FK-506 is required for the CRBA carried out in accordance with this invention. Native FK-506 is available from Fujisawa Pharmaceutical Co., Osaka, Japan and is soluble in aqueous amphipathic solvents, e.g., aqueous methanol. [³H]-dihydro FK-506 can be prepared by exposure of native FK-506 to tritium gas in the presence of a reducing agent such as tris

(triphenyl-phosphine) rhodium I chloride, followed by purification by normal phase and reverse phase

chromatography (Amersham Corp., Arlington Heights, IL). Purification can be determined by TLC system. One preparation of [³H]-dihydro FK-506, which was 98+% pure by three different TLC systems, had a specific activity of 51 Ci/mmol or 63.2 m Ci/mg. The process adds one hydrogen and one tritium atom to the molecule. [Mebmt- β-³H]cyclosporine A (³H-CsA) is obtainable from Amersham Co., at a specific activity at 5 to 20 Ci/mmol.

K-506 can also be labeled with γ-ray emitting ¹²⁵I by brief reduction by chloramine-T in the presence of Na¹²⁵I. ¹²⁵I-labeled histamine-FK-506 can be prepared by the method of Wong, et al.²⁴

For fluorescence polarization detection methods suitable for use with, for example, the Abbott

Laboratories, Inc. (Abbott Park, IL) TDX instrument, fluorophore-labeled FK-506 may be used. FK-506 may be labeled with a fluorophore by art-recognized methods.²⁵ Suitable fluorophores include fluorescein, europium and luciferin.

Chemiluminescent labels such as the water-soluble 1,2-dioxetane derivatives that release light energy upon cleavage with a hydrolytic enzyme^26,27 can be obtained from Tropix, Inc., Bedford, MA.

Chemiluminescence may also be the detection method when the FK-506 label is an enzyme that releases light upon addition of a substrate such as the aforementioned 1,2-dioxetanes.

The CRBA carried out in accordance with this invention can be performed by either solution phase or solid phase methods. The principle underlying both methods is the same. Briefly, a competition

equilibrium is set up between a tracer amount of labeled FK-506 and unknown samples containing analyte FK-506 for binding to a fixed amount of the FK-506 receptor protein described above. Following attainment of equilibrium, the amount of labeled FK-506 bound to the receptor protein is determined. The amount of label bound to the receptor will be reduced in the presence of unlabeled FK-506, and this reduction is proportional to the amount of unlabeled analyte present in the unknown sample. The quantitative relationship between the reduction of receptor-bound label and the concentration of analyte in the unknown sample is determined by reference to a standard curve. To generate a standard curve, a fixed amount of receptor protein is exposed to a fixed tracer amount of labeled FK-506 in the presence of zero-to-supersaturating concentrations of standard FK-506. The supersaturating concentration is, ideally, several orders of magnitude greater than the association constant, Ka, of specific binding, and this fraction ("nonspecific binding," NSB) is assumed to be the same for all ligand

concentrations, as nonspecific binding is assumed to be a linear function of ligand concentration.

For a solution phase standard curve assay,

aliquots of an aqueous alcoholic (e.g., 50% ethanol in water) solution of unlabeled FK-506 are added to glass tubes, and the solvent evaporated, e.g., by a gentle stream of N₂ or in the cold under reduced pressure; the amount of FK-506 delivered ranges between zero and about 100,000 ng. For a solution phase CRBA of FK-506 in blood, whole blood is extracted with an amphipathic organic solvent, such as a lower alkanol (e.g., C₁ to C₆ straight or branched chain, primary, secondary or tertiary alcohol) or acetonitrile. "Amphipathic organic solvent" is intended to mean a liquid organic compound having both hydrophilic and hydrophobic properties. The precipitated proteins are removed by, e.g., centrifugation, and the extract containing FK-506 taken to dryness as described below. The residue to be analyzed is taken up in a small volume of a

water/amphipathic organic solvent mixture, e.g., 1:3, and transferred to a reaction tube. To each tube is added a fixed tracer amount (e.g., 0.5 nM, 50,000- 100,000 CPM) of labeled FK-506, e.g., [³H]-dihydro

FK-506, in a small volume (e.g., about 50 μl) of stock solution. To each tube is then added a solution of receptor protein in a small volume, e.g., about

100 μl-200 μl, of binding buffer, and the tubes

incubated until equilibrium or steady state binding is reached, typically about 20 to 90 mins . , at a slightly elevated temperature such as about 30°C to about 40°C. The composition of the binding buffer is not critical. A preferred binding buffer is 20 mM Tris Buffer, pH 7.2, containing 5 mM 2-mercaptoethanol, 0.05% NaN₃ and 7.5% (v/v) fetal calf serum. To determine non-specific binding (NSB), one set of tubes contains a large molar excess of unlabeled ligand, such as a 200-fold molar excess of unlabeled FK-506 delivered in a small volume, e.g., 50 μl.

At the end of the reaction period, for detection methods other than fluorescence polarization, it is necessary to separate protein-bound from free labeled

FK-506. Among the preferred methods in accordance with this invention are:

A. The contents of the reaction mixture are diluted with ice-cold buffer, preferably at neutral pH, the contents filtered through a glass fiber filter such as Whatman GF/B (Whatman Paper, Maidstone, England), and the filter washed with ice-cold buffer. The membrane retains the receptor-bound labeled FK-506 compound. B. This method is analogous to that of A, except that filtration is carried out on a microporous filter such as 0.22 μm nitrocellulose (Millipore Corp., Bedford, MA) prewashed with a solution of an inert protein, e.g., BSA or γ-globulin, to block

nonspecific sites. The filter retains the

receptor-bound labeled FK-506.

C. Following dilution of the reaction mixture with cold buffer, a suspension of polyethyleneglycol (MW 5,000 to 20,000), e.g., 1 ml of a 30 mg/ml suspension, plus a solution of a carrier protein, preferably delivering about 1 mg of carrier BSA or γ-globulin, are added, and the resulting

suspension is mixed. The particles are collected by centrifugation; the pellet contains receptor- bound labeled FK-506. D. Following dilution of the reaction mixture with cold buffer, a suspension of charcoal particles coated with a carbohydrate (e.g., dextran) or carrier protein (e.g., albumin or γ-globulin) is added to the tube, the suspension mixed

thoroughly, then centrifuged in the cold to sediment the charcoal particles. The supernatant fluid will contain the receptor protein-bound labeled FK-506.

E. The reaction mixture, typically a 100 μl aliquot run in duplicate, is poured onto a column of convenient dimension, such as 0.8 x 7.0 cm of a molecular sieve matrix such as LH-20 Sephadex (Pharmacia Fine Chemicals, Piscataway, NJ).

Washing the column with a small volume (e.g., about 0.5 ml of a buffer (e.g., phosphate-buffered saline, pH 7.4), will elute in the void volume, e.g., the first 2 ml, the receptor-bound labeled FK-506. LH-20 is a weakly hydrophobic matrix, and free FK-506 or CsA will be retarded in such a matrix.

Where a β-emitting radioactive tracer is employed with Methods A and B, each filter is placed in a LSC vial, an aqueous-organic solvent phase combining scintillation system (e.g., PCSS, Amersham, Arlington Heights, IL) is added, and the amount of radioactivity quantified. With Methods C and D, the pellet is suspended in scintillant solution (e.g., PCSS) or dissolved in NaOH and diluted with scintillant

solution, and then counted. With Method E, an aliquot of the void volume is diluted in scintillant solution and counted. In all methods, when ¹²⁵I is the tracer, filters, pellets or solutions containing labeled FK-506 are placed in counting tubes and counted in a

γ-counter.

For quantification of labeled FK-506 by

chemiluminescence when the reporter molecule is a chemiluminescent 1,2-dioxetane such as AMPPD or AMPGD (Tropix, Inc., Bedford, MA) with methods A and B filters are placed on a sheet of blotting paper, and the filters soaked with a solution of the enzyme, e.g., alkaline phosphatase or galactosidase, that hydrolyses the 1,2-dioxetane and produces light. Filters are transferred to a piece of polyester film (e.g.. Mylar), and then to a black box containing instant film, such as Type 612 Polaroid film. After exposure of the film to the emitted light, the dark image is digitized using e.g., a black and white RBP Densitometer, Tobias,

Assoc, Inc., Ivyland, PA. In Method C, the pellet is suspended in a buffer (pH 7-12) containing the

appropriate enzyme and cofactors until maximum

luminescence is attained, typically in 15-30 mins. at

30°C, and the luminescence read in a luminometer, e.g., Turner 20E or Berthold Clinilumat instruments. In Method D, the void volume is reacted with an

appropriate hydrolytic enzyme until maximum

luminescence is attained, typically in 15 to 30 mins. at 30°C, and the luminescence quantified in a

luminometer. The methods are the same when FK-506 is labeled with the enzyme, such as alkaline phosphatase or α - or β-galactosidase except that the appropriate chemiluminescent substrate (AMPPD and AMPGD,

respectively) is added to initiate the production of light.

The principle underlying fluorescence

polarization-based assays is described by Robbins, et al.²⁸ and in the Abbott Laboratories 55 TDX Instruction Manual. In a fluorescence polarization CRBA carried out in accordance with this invention, fluorescein-FK- 506 will not produce a polarized fluorescence signal as this molecule rotates freely, whereas the same molecule bound by a FK-506 receptor protein will produce a signal as it is not free or as free, to rotate. Thus, receptor-bound and free fluorescein-FK-506 do not need to be physically separated in order to carry out this type of assay.

The assay system thus involves carrying out a CRBA with incubation of an initial sample containing

standard or unknown FK-506, labeled FK-506 (e.g., fluorescein-FK-506), and water-soluble receptor

protein. The polarization intensity of the signal is inversely related to analyte concentration.²⁹

Therefore, a patient sample containing a low

concentration of FK-506 analyte will, after equilibrium has been reached in the CPBA of this invention, have a high concentration of receptor protein bound tracer in the reaction mixture, and polarization will be high.

The fluorescence polarization CRBA for FK-506 carried out in accordance with this invention is readily adaptable to the Abbott Laboratories TDX

System. In this adaptation, FK-506 standards,

controls, and patient samples are placed in individual cartridges of the TDX instrument. A Metabolite Reagent Pack containing, in separate vials, a buffer-surfactant solution, a solution of FK-506 receptor protein containing a protein stabilizer, fluorophore-labeled- FK-506 in a solution containing a surfactant and protein stabilizer, is placed in the instrument.

Thereafter, in an automated series of steps, test samples are mixed with receptor protein and

fluorescein-FK-506, and the mixtures are incubated at 37°C for a selected period until equilibrium binding is reached . Thereafter, samples are transferred to glass cuvettes, and the fluorescence polarization signal measured.

For presentation of CRBA data in accordance with this invention, standard curves are drawn for known FK- 506 standard solutions by plotting

[Bound_(std) - NSB/Bound_{(o std)} - NSB]100 vs log[FK-506] wherein Bound_(std) is the total amount of labeled FK-506 bound at each concentration of standard FK-506,

Bound_{(o std)} is the amount of labeled FK-506 bound in the absence of standard FK-506 , and NSB represents

nonspecific binding at each concentration of standard FK-506.

Thereafter, radioactivity, fluorescence

polarization or chemiluminescence values for unknowns and controls are converted to

[Bound_(unk)/Bound_(o)]100 wherein Bound_(unk) is the quantitative value of receptor- bound and Bound(o) is the appropriate control value, by standard calculations. The calculated ratio is then referenced to the standard curve for estimation of the concentration of FK-506 or FK-506-like molecules in the unknown samples. The CRBA of the invention can also be carried out in a solid state system. A supporting matrix, e.g., the bottom of wells of a microtitre plate or the walls of a tube or plastic beads is coated with FK-506 receptor protein, and NSB sites are blocked by brief exposure to an inert protein, e.g., drug-free serum or serum albumin. An aliquot of a solution of labeled FK-506 is contacted with the coated surface with gentle shaking, and the solid surface washed with cold buffer solution, e.g., PBS at ice-bath temperatures.

Thereafter, an aliquot of a patient sample containing FK-506 , its metabolites, or derivatives or analogues is contacted with a receptor protein-coated surface with gentle shaking for a suitable period, e.g., 0 hrs. (control) to 16 hrs (analyte) in the cold. When equilibrium binding has been reached, the incubation fluid is removed, and the solid surface washed gently with cold buffer solution.

Protein-bound labeled FK-506 is removed from the solid surface by a surfactant solution or an alcohol, and the precipitated proteins removed by brief

centrifugation. Thereafter the amount of label is quantified as described above for radioactively-labeled or chemiluminescence-labeled FK-506. The calculations for standards and unknowns are carried out as above.

In order that those skilled in the art can more- fully understand this invention, the following examples are set forth. These examples are given solely for illustrative purposes, and should not be considered as expressing limitations unless so set forth in the appended claims. EXAMPLE 1

Binding of CBA and FK-506 to Cytosolic Protein

Binding of ³H-CsA to proteins in a 100,000 x g cytosol derived from CEM cells was determined using Method E (LH-20) for separating protein bound from unbound ligands.

Whole cytosol (1 mg protein/ml) was incubated with [³H]-CsA (60,000 cpm) in the absence and presence of unlabeled CsA and FK-506 (each 10 μg/ml). Unlabeled CsA, but not FK-506, competed against labeled CsA for binding to cytosolic proteins (Fig. 1).

EXAMPLE 2

A Specific Binding Protein for FK-506 in CEM Cytosol

Cytosol from CEM cells (1 mg protein/ml) was incubated with ³H-FK-506 (60,000 cpm) in the absence and presence of unlabeled FK-506 (10 μg/ml), and protein- bound and free labeled FK-506 separated by the Method E (LH-20). The results (Fig. 2) show that crude cytosol from CEM lymphocytes contains a specific binding

protein for FK-506.

EXAMPLE 3

Purification of a FK-506

Binding Protein from CEM Cytosol

CEM cytosol samples were filtered and injected using either a 250 μl or 500 μl loop with a Beckman

HPLC instrument using a 7.5 x 300 mm Bio-Rad BioSil SEC 125 TSK column, a buffer consisting of 20 mM sodium phosphate, pH 6.8, and a flow rate of 1.0 ml/min.

Fractions were collected, pooled, concentrated by rotary evaporation, assayed for protein by the BCA method, and assayed for binding to ³H-CsA (C-CsA in Fig. 3) or [³H]-dihydro FK-506 (C-506 in Fig. 3). While CsA bound to fractions representing protein molecular weights of about 150 kDa, 50 kDa and 17 kDa, FK-506 bound to only a single peak at about 8-10 kDa. The specific binding activity for FK-506 in the 8-10 kDa fraction was substantially greater than that for CsA in any of its protein fractions (Fig. 3).

EXAMPLE 4

Direct Binding Profile for FK-506 in JURKAT Cytosol

The JURKAT cell line was maintained in RPMI 1640 medium (MA Bioproducts, Walkersville, MD) with 10% FCS supplemented with L-glutamine and antibiotics (Life Technologies, Gaithersburg, MD) at 37°C and 5% CO₂.

Cells (5 x 10⁹) were collected, pelleted and washed with medium three times, and either used immediately as a source for cytosolic proteins or were frozen as a cell pellet at -70°C. Cells for extraction of proteins were homogenized with either a Teflon or ground glass homogenizer, using 0.02 M sodium phosphate pH 6.8 with 0.5% sodium azide on ice. Completeness of cellular disruption was monitored with trypan blue exclusion. The crude homogenates were spun at 100,00 x g for

1 hour, using a Beckman L5-65 ultracentrifuge and a 40.1 rotor and the supernatant fluid (S-100) isolated. The S-100 supernatant fluid (cytosol) was removed and the pellet discarded. Protein determinations were performed using either the Bradford procedure³⁰ or binding to Coomasie blue using a Cobas Bios instrument (Roche Co., Nutley, NJ). The S-100 supernatants were aliquoted and stored at -70°C.

A sample of cytosol derived from JURKAT

lymphocytes was equilibrated with [³H]-dihydro FK-506 or CsA(11.8 mol), and the solution passed through a molecular weight calibrated Bio-Rad Bio-Sil SEC 125 HPLC column at 1.0 ml/min. Fractions were collected and counted by liquid scintillation spectrometry in Packard Gold scintillation fluid. The binding profile shown in Fig. 4 demonstrates that FK-506 was bound primarily to a protein eluting in the 8-10 kDa

molecular weight region, although FK-506 binding proteins also eluted in the 50 kDa region. CsA binding proteins eluted primarily in two molecular weight ranges, one at about 15-17 kDa (cyclophilin) and the other at about 51 kDa; no significant binding of CsA occurred in the 8-10 kDa range.

EXAMPLE 5

Purification of a Fk-506 Binding

Protein from JURKAT Cytosol

A. JURKAT S-100 cytosol was prepared as in Example 4.

B. Matrex Gel Blue A Affinity Purification. One ml of the S-100 cytosol (40-55 mg/ml from Example 4) was loaded onto a Matrex gel Blue A column (Amicon Corp., Danvers MA) which was equilibrated with 10 mM sodium phosphate buffer with 0.1% sodium azide pH 6.8. The columns were 0.8 cm x 7.0 cm Kontes disposable columns with a total bed volume of three mis (Kontes, Vineland, NJ) . A step gradient of sodium phosphate buffer, consisting of three mis each (10, 50, 100, 200, 300 and 400 mM) was run through the column and collected into 12 x 75 mm glass tubes. These fractions were assayed for binding, concentrated by rotary evaporation, dialyzed against 0.02 M sodium phosphate buffer, pH 6.8, and protein concentrations were determined as described above. This step readily separates FK-506 receptor protein from CsA binding protein because of large differences in salt concentration needed to elute each protein from this column matrix. As shown in the histogram of Fig. 5, the bulk of the FK-506 receptor protein eluted at 50 mM salt, whereas the major CsA binding protein eluted at about 300 mM salt. This step was also effective in removing the bulk of the undesired protein; from a total load of about 50 mg/ml, the pooled fractions representing the two binding proteins constituted only about 10 mg.

The about 50 kDa protein preparation was also separated using Matrex Gel Blue A resin. As the about 50 kDa proteins did not bind to this matrix, it was used as a negative selection step. Pools of size excluded S-100 samples (about 50 kDa) were concentrated with a 30 kDa cut-off filter

(Centricon, Amicon, Danvers, MA), adjusted to 80 mM sodium phosphate, pH 6.8, and mixed with

0.5 ml of Matrex Gel Blue A for 15 mins. at room temperature. The resin was spun down in a Beckman microfuge, and the supernatant fluid (containing the about 50 kDa proteins) retained. C. Molecular Weight Sieving. Concentrated fractions from Matrex Blue A chromatography (B above) (5-10 mg/ml) enriched for either cyclophilin (about 17 kDa), about 50 kDa binding protein or FK-506 receptor protein (about 8-12 kDa) were filtered through a 0.45 μ filter. Sample volumes of 500 μl were injected into a Beckman System Gold

Programmable Solvent Module #126 HPLC utilizing a Biosil SEC 125 600 x 7.5 mm column (Biorad, Richmond, CA). Buffer consisted of 0.02 M sodium phosphate with 0.1% sodium azide, pH 6.8, at a flow rate of 1.0 ml/minute. Sample elution was monitored at 280 nm with a Beckman Model #160 Absorbance Detector and Model #427 Integrator. Fractions were collected in 12 x 75 mm glass tubes, tested for binding, pooled, concentrated via rotary evaporation and dialyzed against 5 mM potassium phosphate, 0.5% sodium azide at pH 6.8. D. Weak Cation Exchange Separation. Molecular weight purified pools from C above (100-300 μg/ml) were filtered as above and 250 μl were injected into a Beckman TSK CM-2SW Spherogel column (4.6 mm x 26 cm) and collected at a flow rate of 0.5 ml/minute with 5 mM potassium phosphate, 0.5% sodium azide at pH 6.8. The fractions were assayed for

binding, pooled and concentrated by rotary

evaporation.

When the cyclophilin-sized fraction (about 17 kDa) was separated by weak cation exchange

chromatography, only one protein peak eluted (at 9-10 mins.), and this peak corresponded to all of the ³H-CsA binding activity (Fig. 7).

When the FK-506 receptor fraction (about 8-12 kDa) was separated by the same column

conditions, a single protein peak resulted at 7-8 mins., and all of the ³H-FK-506 binding activity eluted with this peak (Fig. 8).

The about 50 kDa fraction from C, above, produced a single binding peak, coincident with a protein peak (12-13 minutes), when chromatographed on a weak cation exchange column (Fig. 9). E. SDS-Polyacrylamide Gel Electrophoresis. Samples for electrophoresis were prepared and separated as described by Laemlli.³¹ Briefly, protein samples (3-5 μg) pooled and concentrated from molecular weight exclusion or cation exchange eluates , were mixed 1:1 with sample preparation buffer (0.125 M Tris pH 6.8, 3% SDS 20% glycerol and 10%

2-mercaptoethanol) and boiled for 3 minutes.

Samples were loaded onto a 10-20% gradient gel (Integrated Separation Sciences, Boston, MA) and were separated at constant current (30 mA). The gel was fixed and stained using the Daichii silver staining procedure (Integrated Separation

Sciences, Boston, MA). The results are shown in Fig. 10.

Lane 1 contains the molecular weight standards. Lane 2 contains the about 8-12 kDa JURKAT FK-506 receptor protein purified through molecule weight exclusion (step C, above), and Lane 3 the same material purified through weak cation exchange (Step D, above). It is clear that, by this criterion, the FK-506 receptor protein of about 8-12 kDa has been purified to homogeneity. Table 1 summarizes the purification data for cyclophilin. Table 2 that for the about 8-12 kDa FK-506 receptor protein, and Table 3 that for the about 50 kDa binding protein.

The data of Table 2 reveals that the FK-506 receptor protein obtained after cation exchange

chromatography had been purified 1, 250-fold over the S-100 material. As shown by the data of Table 1, the same purification scheme purified cyclophilin about 370-fold over the S-100 material. As shown by the data of Table 3, the about 50 kDa binding protein was purified only about 50-fold by the entire purification procedure, a reflection, perhaps, of the higher binding capacity of the starting materials (S-100 cytosol).

EXAMPLE 6

Scatchard Analyses of Binding Proteins

The binding proteins purified in Example 5 were incubated with increasing amounts of labeled ligand and the binding data analyzed according to Scatchard.³² Scatchard plots are shown in Figs. 11A-C.

Fig. 11A represents the direct binding of labeled CsA to the about 50 kDa protein preparation. The Kd of this interaction was about 64 nM, with a binding capacity of about 3.2 nmoles/mg protein. Cooperative binding was not observed.

Fig. 11B represents the direct binding of labeled FK-506 to the same about 50 kDa protein preparation. This interaction yielded a Kd of about 2 nM and a binding capacity of about 0.3 nmoles/mg protein.

When the binding of labeled FK-506 to its major receptor protein (about 8-12 kDa) was analyzed, the Kd was about 1.3 nM and the binding capacity was about 0.5 nmoles/mg protein.

No cooperativity was observed for the interaction of FK-506 with either of its binding proteins.

EXAMPLE 7

Receptor Binding Assay for FK-506 in Whole Blood

Binding Assay

Binding of ³H-FK-506 to the purified about 8-12 kDa JURKAT cell receptor protein from Example 5, Step C, was performed as in Example 2, wherein receptor-bound FK-506 was separated by the LH-20 method [cf.,

separation Method E of the specification]. The Binding Buffer consisted of 20 mM Tris buffer, pH 7.2,

containing 5 mM 2-mercapto-ethanol, 0.05% NaN₃ and 7.5% FCS. Receptor solutions (100 μl of 15 μg/ml protein) were mixed with 50 μl of ³H-FK-506 (about 2 nM, 25,000 CPM) plus 0 to 70 μg/ml unlabeled FK-506 in 12 x 75 mm glass tubes for 20 mins. at room temperature, then placed in ice. Bound and free ligand were separated by loading 100 μl of the cold reaction mixture onto a 7.0 cm x 0.8 cm column of LH-20 equilibrated in Binding Buffer. The first 2 ml of eluate, containing receptor- bound ligand, were collected and placed in

scintillation vials for counting. The CPM were

corrected back to the total volume of reaction mixture and expressed as the mean of at least two replicates. The thus-obtained standard curve is shown in Fig. 12A.

Assay of FK-506 in Whole Blood

Unlabeled FK-506 was added to whole human blood in the concentration range of 0.5 to 10 ng/ml. FK-506 was extracted from blood by extracting 1.0 ml aliquots of the blood with 3 ml of acetonitrile (100%) by vigorous mixing for 30 seconds; precipitated proteins were removed by centrifugation at 2000 x g. Supernatant fluids were transferred to 16 x 100 mm test tubes, and the solvent evaporated by a stream of air at 40°C.

Residues were dissolved in 100 μl of Binding buffer, and the competitive receptor binding assay using the about 8-12 kDa protein carried out as above. The results are shown in Fig. 12B.

The assay appears to be useful over the

concentration range of at least 0 to 5.0 ng/ml FK-506 in whole blood (Fig. 12B). It should be noted that this concentration range falls within the first 10% of the standard curve shown in Fig. 12A, suggesting that the useful range for assays of FK-506 in blood will extend beyond that shown by the data in Fig. 12B.

The above discussion of this invention is directed primarily to preferred embodiments and practices thereof. It will be readily apparent to those skilled in the art that further changes and modifications in the actual implementation of the concepts described herein can easily be made without departing from the spirit and scope of the invention as defined by the following claims.

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Claims (55)

We claim:

1. A purified FK-506 receptor protein for use in a receptor binding assay for FK-506 prepared by a process comprising the steps of:

(a) centrifuging homogenized, disrupted, normal or transformed mammalian cells to produce water- soluble proteins;

(b) fractionating said water-soluble

proteins;

(c) testing the thus-obtained protein

fractions for FK-506 receptor activity; and

(d) selecting for said receptor binding assay those protein fractions having acceptable FK-506 receptor activity.

2. The receptor protein of claim 1, wherein said mammalian cells comprise lymphocytes.

3. The receptor protein of claim 2, wherein said lymphocytes comprise peripheral blood lymphocytes.

4. The receptor protein of claim 3 wherein said lymphocytes comprise tissue culture lymphocytes.

5. The receptor protein of claim 1, wherein said fractionating comprises the steps of passing said water-soluble proteins through:

(a) an affinity chromatography column;

(b) at least one molecular weight exclusion gel column; and

(c) a cation exchange column.

6. The receptor protein of claim 5, further comprising passage through a hydrophobic interaction chromatography column.

7. The receptor protein of claim 1, wherein said fractionating comprises passing said water soluble proteins through an affinity chromatography column.

8. The receptor protein of claim 7, further comprising passage through at least one molecular weight exclusion gel column.

9. The receptor protein of claim 8, further comprising passage through a hydrophobic interaction chromatography column.

10. The receptor protein of claim 1, wherein said fractionating comprises passing said soluble proteins through at least one molecular weight exclusion gel column.

11. The receptor protein of claim 10, further comprising passage through a cation exchange column.

12. The receptor protein of claim 11, further comprising passage through a hydrophobic interaction chromatography column.

13. The receptor protein of claim 1, wherein said fractionating comprises passing said soluble proteins through a cation exchange column.

14. The receptor protein of claim 13, further comprising passage through a hydrophobic interaction chromatography column.

15. The receptor protein of claim 1, wherein said fractionating comprises passing said soluble proteins through a hydrophobic interaction chromatography column.

16. The receptor protein of claim 1, wherein said protein has a molecular weight within the range of about 1,000 Daltons to about 200,000 Daltons.

17. The receptor protein of claim 1, wherein said protein has a molecular weight in the range of about 8,000 Daltons to about 12,000 Daltons.

18. The receptor protein of claim 1, wherein said protein has a molecular weight in the range of about

40,000 Daltons to about 60,000 Daltons.

19. The receptor protein of claim 1, wherein said testing for said acceptable FK-506 receptor activity comprises a competitive binding assay wherein varying amounts of unlabeled FK-506 compete against a fixed tracer concentration of labeled FK-506 for binding to said FK-506 receptor protein.

20. A purified FK-506 receptor protein for use in a receptor binding assay for FK-506 prepared by

recombinant DNA techniques.

21. The receptor protein of claim 20, wherein said protein has a molecular weight within the range of about 1000 Daltons to about 200,000 Daltons.

22. The receptor protein of claim 20, wherein said protein has a molecular weight in the range of about 8,000 Daltons to about 12,000 Daltons.

23. The receptor protein of claim 20, wherein said protein has a molecular weight in the range of about 40,000 Daltons to about 60,000 Daltons.

24. A quantitative receptor assay for FK-506 contained in an initial fluid sample, comprising the steps of:

(a) preparing an extract of said initial fluid sample, said extract containing said FK-506;

(b) contacting said FK-506 in said extract and labeled FK-506 in a tracer concentration with an aqueous solution of a purified FK-506 receptor protein until equilibrium or steady-state binding is reached;

(c) physically separating receptor-bound from unbound labeled tracer FK-506;

(d) quantifying the amount of said labeled tracer FK-506 specifically bound to said FK-506

receptor protein;

(e) determining the amount of FK-506 in said initial fluid sample by referring said quantified amount of said labeled tracer FK-506 specifically bound to said FK-506 receptor protein in step (d) hereinabove to a standard curve prepared by analyzing a series of standard solutions of FK-506 by the competitive

receptor binding assay of steps (a) to (d), inclusive, hereinabove, and plotting [B_(std) - NSB/B_{(o std)} - NSB] vs log [FK] wherein B_(std) is the amount of said labeled tracer bound to said receptor, B_{(o std)} is the control, NSB is the nonspecific binding of sample of said labeled tracer, and [FK] is the concentration of said standard solution of FK-506 assayed.

25. The method of claim 24 , wherein said extract is prepared by contacting said initial fluid sample with an amphipathic organic solvent.

26. The method of claim 24, wherein said label in said labeled tracer FK-506 is radioactivity.

27. The method of claim 26, wherein said

radioactivity is selected from a group consisting of α, β, or γ-ray emitters.

28. The method of claim 24, wherein said label in said labeled tracer FK-506 is a compound capable of producing luminescence.

29. The method of claim 24, wherein said label in said labeled tracer FK-506 is a compound capable of producing fluorescence.

30. The method of claim 24, wherein said label in said labeled tracer FK-506 is a compound capable of producing color.

31. The method of claim 24, wherein said label in said labeled tracer FK-506 is an enzyme.

32. The method of claim 24, wherein said purified FK-506 receptor protein is prepared as claimed in any one of claims 1 to 23 inclusive.

33. The method of claim 24, wherein the method of said physical separating of said receptor bound and unbound labeled tracer FK-506 is selected from a group consisting of filtration through a filter or membrane, charcoal adsorption, protein precipitation or

filtration through a molecular weight exclusion column.

34. The method of claim 24 carried out in a solid state system.

35. The method of claim 34, wherein said purified FK-506 receptor protein is coated on a solid surface, said FK-506-containing extract of said initial fluid sample and said labeled tracer FK-506 are contacted with said receptor protein coat until equilibrium or steady-state binding is reached, the amount of said labeled tracer FK-506 bound to said receptor coat is quantified, and, the amount of FK-506 in said initial fluid sample is quantified by reference to a standard curve prepared by substituting a series of FK-506 standard solutions for said initial fluid sample extract in said receptor binding assay.

36. A quantitative receptor assay for FK-506 contained in an initial fluid sample, comprising the steps of:

(a) preparing an extract of said initial fluid sample, said extract containing said FK-506; (b) contacting said FK-506 in said extract and a fluorophore-labeled FK-506 with an aqueous solution of a purified FK-506 receptor protein until equilibrium or steady state binding is reached;

(c) measuring the fluorescent polarization signal of the thus-incubated mixture, and converting said fluorescent polarization signal to an amount of receptor-bound, fluorophore-labeled FK-506, and;

(d) determining the concentration of FK-506 in said initial fluid sample by reference to a

fluorescence polarization standard curve obtained by analyzing a series of standard solutions of FK-506 by said fluorescence polarization receptor binding assay, and plotting [B_(std)/B_{(o std)}]100 vs log[FK] wherein the terms B_(std)/B_{(o std)} and [FK] are as defined in claim 13.

37. The method of claim 36, wherein said extract is prepared by contacting said initial fluid sample with an amphipathic organic solvent.

38. The method of claim 36, wherein said

fluorophore is selected from a group consisting of fluorescein, luciferin, and europium.

39. The method of claim 36, wherein said purified FK-506 receptor protein is prepared as claimed in any one of claims 1 to 23, inclusive.

40. A quantitative receptor assay for FK-506 contained in an initial fluid sample, comprising the steps of:

(a) preparing an extract of said initial fluid sample, said extract containing said FK-506;

(b) contacting said FK-506 in said extract and an enzyme-labeled FK-506 with an aqueous solution of a purified FK-506 receptor protein until equilibrium or steady-state binding is reached;

(c) physically separating receptor-bound from unbound enzyme-labeled FK-506;

(d) quantifying the amount of said enzyme- labeled FK-506 specifically bound to said receptor protein; and

(e) determining the amount of FK-506 in said initial fluid sample by referring the quantified amount of said enzyme-labeled FK-506 specifically bound to said receptor protein obtained in step (d) hereinabove to a standard curve prepared by analyzing a series of standard solutions of FK-506 by the competitive

receptor binding assay of steps (a) - (d), hereinabove, and plotting

[B_(std) - NSB/B_{(o std)} - NSB]100 vs log [FK] wherein the terms B_(std), NSB, B_{(o std)} and [FK] are as defined in claim 13.

41. The method of claim 40, wherein said extract is prepared by contacting said initial fluid sample with an amphipathic organic solvent.

42. The method of claim 40, wherein said purified FK-506 receptor protein is prepared as claimed in any one of claims 1 to 23, inclusive.

43. The method of claim 40, wherein said

quantifying comprises a chemiluminescent method.

44. The method of claim 43, wherein said

chemiluminescent method comprises decomposing an enzyme-decomposable water-soluble chemiluminescent 1,2-dioxetane derivative with said enzyme linked to said enzyme-labeled tracer FK-506 to yield optically- detectable light energy.

45. The method of claim 44, wherein said enzyme is an α- or β-galactosidase and said chemiluminescent substrate is AMPGD.

46. The method of claim 44, wherein said enzyme is a phosphatase and said chemiluminescent substrate is AMPPD.

47. The method of claim 40 carried out in a solid state.

48. A quantitative receptor assay for FK-506 contained in an initial fluid sample, comprising the steps of:

(a) preparing an extract of said initial fluid sample, said extract containing said FK-506;

(b) contacting said extract of said initial fluid sample and a chemiluminescent compound-labeled FK-506 with an aqueous solution of a purified FK-506 receptor protein until equilibrium or steady-state is reached;

(c) physically separating receptor-bound from unbound chemiluminescent compound-labeled FK-506;

(d) quantifying the amount of said FK-506 specifically bound to said receptor; and,

(e) determining the amount of FK-506 in said initial fluid sample by referring the quantified amount of said labeled FK-506 specifically bound to said receptor obtained in step (d) hereinabove to a standard curve prepared by analyzing a series of standard solutions of FK-506 by the competitive receptor binding assay of steps (a) - (d), hereinabove, and plotting

[B_(std) - NSB/B_{(o std)} - NSB]100 vs log [FK] wherein the terms B_(atd), NSB, B_{(o std)} and [FK] are as defined in claim 13.

49. The method of claim 48, wherein said extract is prepared by contacting said initial fluid sample with an amphipathic organic solvent.

50. The method of claim 48, wherein said purified FK-506 receptor protein is prepared as claimed in any one of claims 1 to 23, inclusive.

51. The method of claim 48, wherein said

quantifying comprises contacting said chemiluminescent compound-labeled FK-506 with a hydrolytic enzyme specific for said chemiluminescent compound, and measuring the optically-detectable light energy produced.

52. The method of claim 51, wherein said

chemiluminescent compound is AMPGD and said hydrolytic enzyme is an α- or β-galactosidase.

53. The method of claim 51, wherein said

chemiluminescent compound is AMPPD and said hydrolytic enzyme is a phosphatase.

54. The method of claim 48 carried out in a solid state.

55. A kit for a quantitative receptor assay for FK-506 in an initial fluid sample comprising

(a) purified FK-506 receptor protein as claimed in any one of claims 1 to 23, inclusive;

(b) labeled FK-506; and

(c) FK-506 standards.