CA2286311A1 - Method for production of antibodies to specific sites of rapamycin - Google Patents

Abstract

This invention relates to the production of polyclonal and monoclonal antibodies to specific sites of rapamycin (Sirolimus). The reactivity of these poly and monoclonal antibodies make them particularly useful for immunoassays for therapeutic drug monitoring (TDM). These immunoassays or TDM kits may include polyclonal or monoclonal antibodies to specific sites of rapamycin. These kits may also include various combinations of polyclonal antibodies, polyclonal and monoclonal antibodies or a panel of monoclonal antibodies. Rapamycin conjugate immunogens are prepared for the immunization of a host animal to produce antibodies directed against specific regions of the rapamycin molecule. By determining the specific binding region of particular antibody, immunoassays which are capable of distinguishing between the parent molecule, active metabolites, inactive metabolites and other structurally similar immunosuppressant compounds are developed. The use of divinyl sulfone (DVS) as the linker arm molecule for forming rapamycin-protein conjugate immunogens is described. DVS-linked rapamycin-protein conjugates were found to elicit antibodies with greater specificity to the rapamycin molecule than succinate linked conjugates.

Description

Method for Production of Antibodies to Specific Sites of Ranamycin Introduction and Background This invention relates to the production of polyclonal and monoclonal antibodies to specific sites of rapamycin ISirolimusl. The reactivity of these poly and monoclonal antibodies make them particularly useful for immunoassays for therapeutic drug monitoring (TDM1. These immunoassays or TDM kits may include polycional or monoclonal antibodies to specific sites of rapamycin.
These kits may also include various combinations of polyclonal antibodies, polyclonal and monoclonal antibodies or a panel of monoclonal antibodies.
Rapamycin (Rapa) is a macrocyclic antibiotic, which was originally isolated in soil samples from Easter Island from a Streptomyces hygroscopicus strain.' Rapamycin is structurally related to the immunosuppressant FK-506 (Tacrolimus) but mechanistically different. Rapamycin is also a potent immunosuppressant that inhibits T and B cell activation by blocking cytokine-mediated events, and inhibits growth-factor mediated cell proliferation. The structure of rapamycin is given below:
HO
41~~~4 41 3g~
H 3 C O ~~"w 46 ll , 38 JIP~ 5... ~ ~ "' ~ 27 I
0 ~_:~2~~ 29 3g O H
H3C~ 3 16 0 14 \2 0 / 1~ OH H3c0;' H3C ~3~ OCH3 31 ' 16 /.. , ti / \ 3 \ / 38 36 Structure of Slrollim us (RAPA) Currently, the two most commonly administered immunosuppressive drugs to prevent organ rejection in transplant patients are cyclosporine (CSA) and FK-506 (FK1. Therapeutic monitoring of concentrations of these drugs in blood is required to optimize dosing regimes to ensure maximal immunosuppression with minimal toxicity. Recent clinical data indicates that rapamycin will be a widely used immunosuppressant to prevent organ rejection in transplant patients.

Specific TDM monitoring kits for rapamycin will therefore be required. The polyclonal and monoclonal antibodies to specific sites of rapamycin of this invention are ideally suited for developing rapamycin TDM kits.
Cytochrome P45° 3A4 enzyme metabolizes rapamycin to a number of demethylated and hydroxyiated metabolites. The exact pathways of rapamycin metabolism in humans have not been completely elucidated since only a few of the metabolites have been structurally identified. Therefore, no consensus has been established concerning the identity or steady state concentrations in whole blood after oral administration. A summary of the current reported knowledge of rapamycin metabolism follows.
Streit et. al. structurally identified 'four rapamycin metabolites from rabbit liver microsomes.Z These include 41-deme;thyl rapamycin, 7-demethyl rapamycin, 1 1-hydroxy rapamycin, and a 24-hydroxy .ester hydrolysis degradation product of rapamycin. It has also been shown that the metabolites of rapamycin can undergo this ester hydrolysis. Streit also partially identified di, tri, and tetra hydroxylated rapamycin metabolites. Wang et. a/, found 16 hydroxylated and or demethylated metabolites in the bile of rapamycin treated rats.3 Nickmilder et. al.
identified a 3,4 and 5,6 dihydrodiol rapamycin metabolite in rat liver microsomes.4 In trough whole blood, Streit et. al. have identified 4-1-demethyl, hydroxy, dihydroxy, and didemethyl rapamycin metabolites.5 ThesE; metabolites accounted for 56% of total rapamycin derivatives measured. Finally, l-eung et. al. looked at the disposition of ["C]-rapamycin in healthy male volunteers.fi They found that rapamycin represented approximately 35% of the total radioactivity in blood and that 41 demethyl, 7-demethyl, and several hydroxyr, hydroxydemethyl, and didemethyl rapamycin metabolites individually represented between 1 and 12% of the total radioactivity. They also found there was no notable presence of glucuronide or sulfate conjugates in blood, feces, or urine and that most of an oral dose was eliminated in feces.
Rapamycin metabolites can be isolated from a number of various sources, including but not limited to blood, urine or feces samples, from liver microsomes or from microorganism cultures.
Detailed Description of the Invention Rapamycin conjugate immunogens are prepared for the immunization of a host animal to produce antibodies directed against specific regions of the rapamycin molecule. By determining the specific binding region of particular antibody, immunoassays which are capable of distinguishing between the parent molecule, active metabolites, inactive metabolites and other structurally similar :3 immunosuppressant compounds are developed. The use of divinyl sulfone (DVS) as the linker arm molecule for forming rapamycin-protein conjugate immunogens is described. DVS-linked rapamycin-protein conjugates were found to elicit antibodies with greater specificity to the rapamycin molecule than succinate linked conjugates. The following examples describe the best mode for carrying out the invention.
Example 1 - Synthesis of Rapamycin-42-Divinyl Sulfone and Coniugation to a Protein Carrier Preparation of rapamycin-42 divinyl sulfone hapten: Rapamycin f0.5 mmol) (Calbiochem-Novabiochem, San Diego, Cat. # 553210) was dissolved in dichloromethane and treated with 10 equivalents of 2-t-Boc aminoethylchloroimidate and the reaction mixture is cooled to 0°C. To this solution, 4 mL of trimethylsilyl triflate was added in one addition. The reaction mixture was stirred at 0°C for 24 hours. Then the reaction mixture was diluted with dichloromethane (100 mL) and washed with water (50 mL x 3). The organic solution was dried and concentrated and 'the mixture subjected to column chromatography to remove the excess of chloroimidate reagent. This material was analyzed using MS-flow injection electrospray mass spectrometry. The derivatized rapamycin was then treated with trifluoroacetic acid to remove the amino protecting group. The reaction mixture was then diluted with dichloromethane (50 mL) and washed with water. The organic solution was dried and concentrated to get the aminoethyl derivative of rapamycin. Without further purification, the reaction mixture was treated with an excess of divinylsulfone in dichloromethane solution using anhydrous potassium carbonate as the catalyst. The reaction mixture was stirred for 24 hours and then diluted with dichioromethane and washed with water to remove the carbonate. The organic solution was dried and concentrated and the crude product subjected to column chromatography to remove the excess of divinyl sulfone. The isolated product was used for conjugation without further purification.
Preparation of rapamycin-42 divinyl sulfone conjugate: Conjugation of the rapamycin-42-divinyl sulfone derivative was performed by preparing a solution of the rapamycin-42-divinyl sulfone derivative in dimethyl sulfoxide which was then slowly added to a rapidly stirred solution of keyhole limpet hemocyanin (KLH) or - human serum albumin (HSA) in 0.2M phosphate buffer (pH 7.61. Stirring of the mixture was continued at room temperature for 24 hours followed by isolation of the rapamycin-42-divinyl sulfone protein conjugate by dialysis.

WO 98!45333 PCT/CA98/00361 Example 2 - Synthesis of Rapamycin-42-~;uccinate and Coniugation to a Protein Carrier Preparation of rapamycin-42-O-hemisuccinate: Dimethylaminopyridine (11.8 mg, 97 umol) was added to a solution of rapamycin (80.0 mg, 88 ymoll and succinic anhydride (30.7 mg, 307 pmol) in 2 mL dry pyridine and the mixture stirred at room temperature for 23 hours. The pyridine was evaporated and the residue dissolved in ethyl acetate. The ethyl acetate solution was washed twice with water and finally with brine before drying over magnesium sulfate and evaporating the solvent. The residue was eluted through a silica gel column using methanol/chloroform (1:19) and then metlnanol/chloroform (1:9) as eluent to give 20.0 mg (23%) of the product as a colorlcas solid.
Preparation of 42-O-succinimidooxysuccinyl rapamycin: N-hydroxysuccinimide (2.3 mg, 19.7 ~cmoli was added to a solution of rapamycin-42-O-hemisuccinate (20.0 mg, 19.7 umol) and 1-ethyl-3-(3-dinnethylaminopropyllcarbodiimide (EDAC) (3.8 mg, 19.7~mol) in 5 mL of dry dichloromethane and the mixture stirred overnight at room temperature. The solvent was evaporated and the residue eluted through a silica gel column using ethyl acetate as eluent to give 5.7 mg (26%) of the product as a colorless solid.
Analysis of rapamycin-42-O-hemisuccinatc: and 42-O-succinimidooxysuccinyl rapamycin:
Purified rapamycin-42-O-hemisuccinate (1'013.5 daltons) was identified as the sodium adduct (1036.5 daltons) by electrospray ionization mass spectrometry and structurally characterized by fragmentation in the negative-ion mode. Purified O-succinimidooxysuccinyl rapamycin (1110.5 daltons) was identified as the sodium adduct (1133.5 daltons) by electrospray ionization mass spectrometry.
Preparation of rapamycin-42-O-succinate Conjugates: A solution of 42-O-succinimidooxysuccinyl rapamycin (2.0 mg) in 500 mL of dimethyl sulfoxide was slowly added into a rapidly stirred solution of keyhole limpet hemocyanin (KLH) (3.0 mg) or human serum albumin (HSA) in 2 mL of 0.1 M aqueous sodium bicarbonate adjusted to pH 7.7 with acetic: acid. Stirring of the mixture was continued at room temperature for 24 hours followed by isolation of the rapamycin-42-divinyl sulfone protein conjugate by dialysis.
Example 3 - Synthesis of Rapamycin-27-C)xime-Divinyl Sulfone and Coniuctation to a Protein Carrier:

ci Preparation of rapamycin-27-oxime: Hydroxylamine hydrochloride (3.0 mg, 44 pmol) in 100 mL of water was added to a solution of rapamycin (20.0 mg, 22 Eimol) and pyridine (40 mL) in 4 mL of ethanol and the reaction mixture stirred at room temperature for 24 hours. The reaction mixture was diluted with ethyl acetate and washed sequentially with wai:er, dilute aqueous hydrochloric acid, and brine. The organic phase was dried over magnesium sulfate and the solvent evaporated to give 20 mg of crude product.
Analysis of rapamycin-27-oxime-divinyl: l.C/MS analysis (Gradient conditions:
25/25/50 water/acetonitrile/methanol at 0 minutes up to 20/30150 water/acetonitrile/methanol at 18 minutes. Column: Spherisorb C-8 semi-prep.
Temperature was 35° C and the flow rate set at 3.5 mL/min. The UV
signal was monitored at 276 nm) of the crude residue; indicated that there were two isomeric forms of the oxime as well as a small amount of unreacted rapamycin. Negative-ion fragmentation of Rapa-Oxime is consistent with oxime formation at C-27.
The mixture was used without purification for further reaction.
Preparation of the rapamycin-27-oxime-divinyl sulfone hapten (Rapa-Ox-DVS):
Vinyl sulfone (203 mg, 1.72 umol) was aclded to a mixture of the crude rapamycin-27-oxime (20 mg, 22 Eimol) and dried anhydrous potassium carbonate (80 mg) in 10 mL dry dichloromethane at room temperature and under a nitrogen atmosphere. The mixture was stirred for '17 hours. Passing a stream of nitrogen through the flask evaporated the solvent and the resulting residue was immediately quenched with 10 mL of a sollution of 10 drops acetic acid in 10 mL
methanol. The clear solution was then decanted off from the remaining potassium carbonate granules and the solution concentrated. The residue was passed through a silica gel column using a gradient of methanol/chloroform (1 % to 5%
methanol) as eluent to separate the reaction products from excess vinyl sulfone.
Analysis of rapamycin-27-oxime-divinyl-suilfone hapten (Raga-Ox-DVS1: The crude reaction residue was resolved by reversed-phase HPLC (Gradient conditions:
40/10/50 water/acetonitrile/methanol from 0 to 5 minutes, up to 25/25/50 water/acetonitrile/methanol from 5 to 40 minutes, followed by 50150 acetonitrile/water from 40 to 45 minutes. Column: Spherisorb C-8 semi-prep.
Temperature was 35° C and the flow rate set at 3.5 mL/min. The UV
signal was monitored at 276 nm) into 3 major rapamycin-Ox-DVS species; Rapa-Ox-DVS
/species X); Rapa-Ox-DVS (species 2); Rapa-Ox-DVS (species 3)] which were identified by electrospray ionization mass spectrometry. Under the gradient - conditions specified both Rapa-Ox-DVS (species X) and Rapa-Ox-DVS (species 2) elute as pure product while Rapa-Ox-DV;i (species 3) was further purifed using a 35/15/50 water/acetonitrile/methanol isocyanic mixture and identical chromatographic conditions as above.
The positive-ion fragmentation pattern for Rapa-Ox-DVS (species 2) is consistent with rapamycin modification through the C-27 position. The LC/MS
profile and mass spectrum was obtained for purified Rapa-Ox-DVS (Species 31.
The positive-ion fragmentation pattern for Rapa-Ox-DVS (species 3) was again consistent with rapamycin modification through the C-27 position.
The yields for each species were as follows:
Rapa-Ox-DVS-(X): 2.4 mg (10%;
Rapa-Ox-DVS-(2): 3.4 mg (15%) Rapa-Ox-DVS-(3): 0.5 mg (2%) Preparation of rapamycin-oxime-Divinyl Sulfone Conjugates: A solution of Rapa-OX-DVS (species 2) (0.3 mg) in 300 mL of dimethyl sulfoxide was slowly added into a rapidly stirred solution of keyhole limpet hemocyanin (KLH) f 1 .0 mg) in 1 mL
of 0.2 M phosphate buffer (pH 7.6) and the mixture stirred at room temperature for 24 hours. The reaction mixture was then dialyzed to recover the rapamycin-oxime-divinyl sulfone protein conjugate. A Rapa-Ox-DVS (species2)-HSA
conjugate was prepared in the same manner.
Example 4 - Synthesis of Rapamycin-31-Divinyl Sulfone and Coniugation to a Protein Carrier:
Preparation of a rapamycin-31-divinyl sulfone hapten (Rapa-DVS): Vinyl sulfone (82.6 mg, 0.7 pmol) was added to a mixture of rapamycin (5.0 mg, 5.5 pmol) and dried anhydrous potassium carbonate (30 mg) in 3 mL of dry acetone at room temperature under a nitrogen atmosphere. The mixture was stirred for 19 hours.
Passing a stream of nitrogen through the flask evaporated the solvent and the resulting residue was immediately quenched with 5 mL of a solution of 10 drops acetic acid in 10 mL methanol. The clear solution was then decanted off the potassium carbonate granules and the solution concentrated. The residue was passed through a silica gel column using a gradient of methanol/chloroform (1 % to 2% methanol) as eluent to separate the reaction products from excess vinyl sulfone. The combined reaction products were then purified and analyzed as follows.
Analysis of a rapamycin-31-divinyl sulfone hapten (Raga-DVS): The crude reaction - residue was analyzed by LC/MS (Gradient conditions: 25/25/50 water/acetonitrile/methanol at 0 minutes up to 20/30/50 water/acetonitrile/methanol at 18 minutes. Column: Spherisorb C-8 semi-prep.
Temperature was 35° C and the flow rate set at 3.5 mL/min. The UV
signal was monitored at 276 nm) and found to contain 1 major species of Rapa-DVS along with its isomer. Rapa-DVS was purified using a isocyanic mobile phase of 40/10/50 water/acetonitrile/methanol ;containing 10% Tert-butyl methyl ether) and identical chromatographic conditions as above. The LC/MS profile and mass spectrum of purified Rapa-DVS was obtained. The positive-ion fragmentation pattern for Rapa-DVS is consistent with rapamycin modification through the 31-OH position. The obtained yield was 0.1 mg (2%) Preparation of rapamycin-31-divinylsulfone conjugates: Rapa-31-DVS-KLH and HSA conjugates were prepared as described in example 3.
Example 5 - Isolation and Characterization of Raoamycin Metabolites:
Biosynthesis of Rapamvcin Metabolites Utilizing Rabbit Liver Microsomes:
A Biosynthesis The basic procedure utilized for isolating rapamycin metabolites was as follows:
1 . Preparation of Rabbit Liver Microsomes A fresh or frozen rabbit liver (not induced) is washed with approximately 750 mL of 1.15% KCI (w/v) and cut into small pieces (approximately 5 mm3). These are placed into a small conical 50 mL centrifuge tube with 15 mL of 1.15% KCI
and stored on ice. After the whole liver has been processed, the pieces are homogenized using a Beckman Ploytron homogenizer into a microsomal suspension that is centrifuged at 10,000 xg for 20 min. Following centrifugation the supernatant is decanted into specialized centrifuge tubes and placed on ice.
These are centrifuged again, using an ultracentrifuge, for 60 min at 100,000 xg.
This process will yield a microsomal pellet vvhich contains the cytochrome P45° enzymes required for the metabolism of rapamycin. The microsomes are then re-suspended in 1.15% KCI, tested for protein concentration using the Lowry method, and stored at -70°C.
2. Biosynthesis of Rapamycin Metabolites Incubation mixtures have a final volume of 45 mL and will contain 22.5 mg of rapamycin dissolved in 1.8 mL DMSO, The reaction mixture will also contain 0.1 SUBSTITUTE SHEET (RULE 26) M sodium phosphate buffer (pH 7.4), 0.5 mM EDTA, 5.0 mM MgCl2, 3.5 mM
NADPH, 1 .5 mM NADP, 50 mM glucose-6-phosphate, 10 units per mL of glucose-6-phosphate dehydrogenase, and 10 mg/mL of microsomal protein.
The biotransformation reaction is carried out in 250 mL Erlenmeyer flasks.
The microsomal solution, without drug, is allowed to incubate at 37°C
for 5 min in an environmentally controlled incubator shaker. The reaction is initiated by adding the drug and allowing the reaction to proceed for two hours. At this time, the reaction is stopped by removing the flasks from the incubator, transferring their contents into 50 mL centrifuge tubes, and storing them at -20°C.
3. Metabolite Isolation The metabolites are isolated by thawing the stored reaction mixtures and transferring them to 500 mL glass booties (100 mL of reaction mixture per bottlel.
This solution is acidified with an equal volume of 0.2 M acetic acid IpH 3.0) and extracted two times with 200 mL MTBE (methyl tert butyl ether). The solvent is recovered and evaporated to dryness using a rotary evaporator. The residue is reconstituted in methanol and stored at -70°C.
4. Metabolite Purification A Waters chromatographic system comprised of a 600E gradient controller plus pump, 717 autos ampler, 486 U'J detector, and Millenium workstation was used to separate and purify the rapamycin metabolites. The column utilized for initial separation is a Waters C8 reverse phase (10 x 250 mm) Spherisorb semi prep HPLC column. The metabolites were separated using a column temperature of 60°C and a flow of 2.5 mL/min. The initial mobile phase consisted of 40%
water and 60% methanol. To achieve: the best separation, this composition was programmed to change over 50 min as indicated in the following table:
Time Flow Water Acetonitrile Methanol Comment lmin)ImL/min)1%) 1%) 1%) 0.00 2.5 40 0 60 gradient 40.002.5 20 60 20 40.012.5 20 40 40 wash 50.00 2.5 40 0 60 equilibrate Individual peaks were collected, pooled, and labeled. Each of these peaks represents a rapamycin metabolite(s). Using the same chromatograhic system, the peaks collected are subjected to further purification using a Waters C18 reverse phase (3.6 x 150 mm) Symmetry column. The column temperature utilized was 60°C, the flow was 1.0 mLlmin, and the mobile phase consisted of a water/
methanol gradient that was specific for each metabolite purified.
Rapamycin Metabolite Species Identified from Microsome Preparations:Raaamycin Metabolite Species Identified from Microsome PreparationsRapamycin Metabolite SUBSTITUTE SHEET (RULE 26) SnPriac Irf~ntifiarf frnm Mir~rnSnmP Prenar.ations:
Molecular Species Peak DesignationMetabolite Adduct Designation ~I Mass 922 7-O-Demethyl Rapa D4 M3 922 41-0-Demethyl RapaF1 M4 922 32-0-Demethyl Rapa 908 32,41-Didemethyl C5 M5 Rapa 908 Didemethyl Rapa 952 C9-C23 Hydroxy D1 M1 Rapa 952 C1-C8 or C31-C36 D3 M2 Hydroxy Ra a 952 Hydroxy Rapa 938 41-O-Demethyl, C 1 C'I -C8 or C31-C36 H drox Ra a 894 7,32,41 Tridemethyl Rapa 954 Demethyl, DihydroxyA8 Rapa or Ester H drol sis 970 Demethyl, TrihydroxyA3 +A4 Rapa or Dih drodiol R a 968 Dihydroxy Rapa B or C

Example 6 - Immunization to Elicit Rapamycin specific Antibody Responses The basic immunization protocols are as follows:
Typically, mice are immunized on day 0 (1 ° - primary immunization), day 7 (2° -secondary immunization), and day 28 (3° - tertiary immunization) by subcutaneous or intraperitoneal injection with rapamycin - conjugate immunogens at doses of 5, 10, 15, or 20 pg based on protein content. Mice were bled 7-10 days post 2° and 3° immunization to collect serum to assay antibody responses. Various other immunization schedules are effective, including day 0 (1 °), day 7 (2°) and days 14, 21 or 30 (3°); day 0 (1 °), day 14 (2°), and days 28 or 44 (3°); and day 0 (1 °), day 30 (2°1 and day 60 (3°), Thirty days post-tertiary immunization a booster may be injected, subsequent monthly boosters may be administered.
Immunized mice are I.V. or I.P. injected with immunogen in PBS as a final boost 3-5 days before the fusion procedure. This increases the sensitization and number of immunogen specific B-lymphocytes in the spleen Ior lymph node tissues). This final boost is administered 2 to 3 weeks after the previous injection to allow circulating antibody levels to drop off.
Such immunization schedules are useful to immunize mice with rapamycin SUBSTITUTE SHEET (RULE 26) immunogen conjugates to elicit specific polyclonal antiserum and for the preparation of specific monoclonal antibodies. The immunogen compositions are also useful for immunizing any animal capable of eliciting rapamycin specific antibodies, such as bovine, ovine, caprine, equine, leporine, porcine, canine, feline 5 and avian and simian species. Both domestic and wild animals may be immunized.
The route of administration may be any convenient route, and may vary depending on the animal to be immunized, and other factors. Parenteral administration, such as subcutaneous, intramuscular, intraperitoneal or intravenous administration, is preferred. Oral or nasal administration may also be used, 10 including oral dosage forms, which are enteric, coated.
Exact formulation of the compositions will depend on the species to be immunized and the route of administration. The immunogens of the invention can be injected in solutions such as 0.9 % NaCI (w/v), PBS or tissue culture media or in various adjuvant formulations. Such adjuvants could include, but are not limited to, Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide, dimethyldioctadecylammonium bromide, Adjuvax (Alpha-Beta Technology), Imject Alum (Pierce), Monophosphoryl Lipid A (Ribi Immunochem Research), Titermax (CytRxl, toxins, toxoids, glycoproteins, lipids, glycolipids, bacterial cell walls, subunits (bacterial or viral), carbohydrate moieties (mono-, di-, tri-, tetra-, oligo- and polysaccharide), dextran sulfate, various liposome formulations or saponins. Combinations of various adjuvants may be used with the immunogen conjugates of the invention to prepare a pharmaceutical composition.
The conjugates of this invention may be used as immunogens to elicit rapamycin or rapamycin metabolite specific poiyclonal antibody, and to stimulate B-cells for specific monoclonal antibody production. They may also be utilized as development and/or research tools; as diagnostic reagents in immunoassay kit development; as prophylactic agents, for example, to block cell receptors; and as therapeutic modalities as immunomodulators and as drug delivery compositions.
Example 7 - Assays to Determine Antibody Reactivity to Rapamycin Immunogens:
The basic direct EL1SA protocol for determining antibody reactivity to rapamycin used in the invention was as follows:
Direct ELISA Protocol:Direct ELISA ProtocolDirect ELISA Protocol:
1 . Use Falcon Pro-bind immunoplate.
2. Dilute coating antigen to 1 .0 pg/mL in carbonate-bicarbonate buffer. Use glass tubes.
3. Add 100 pL to each well of plate. Store overnight at 4° C.

4. Shake out wells and wash 3x with 200 ~cL PBS / 0.05 % Tween (v/v} per well.
5. Add blocking buffer, 100 uL per well 2 % BSA in PBS (w/v). Incubate for 60 min at 37° C.

6. Wash 3x as in step 4.

7. Add 100 pL per well of test antibody appropriately diluted in PBS / 0.1 Tween (v/v). Incubate 60 min at 37° C.

8. Wash 3x as in step 4.

9. Dilute alkaline phosphatase conjugated anti-mouse IgG (Tago cat # AMI 4405) in PBS / 0.1 % Tween to 1 :2000 concentration. Add 100 pL per well and incubate at 37° C for 60 min.

10. Wash 3x as in step 4.
1 1 . Prepare enzyme substrate using Sigma #104 alkaline phosphatase substrate tablets (1 mglmL in 10 % diethanolarnine substrate buffer (v/v)}. Add 100 pL
per well and incubate in the dark at room temperature. Absorbance can be read at 405 nm at approximately 15-min intervals.
To measure antibody isotype levels IIgM, IgG and IgA isotypes} elicited to rapamycin immunogens the following basic procedure was used:
Isotyping ELISA Protocol:
1 . Use Falcon Pro-bind immunoplates.
2. Dilute coating antigen to 1 pg/mL in carbonate-bicarbonate buffer. Add 100 pL per well and incubate overnight at 4° C.
3. Shake out wells and wash 3x with 2C>0 ~L PBS / 0.05 % Tween per well.
4. Add 200 pL blocking buffer per well (PBS / 2 % BSA). Incubate 60 min at room temperature.
5. Wash as in step 3.
6. Add 100 pL per well of tissue culture supernatant undiluted or mouse serum diluted to 1 /100 in PBS / 0.1 % Twee:n. Incubate for 60 min at 37° C.
7. Wash as in step 3.
8. Prepare 1:2 dilution of EIA grade mouse type (rabbit anti-mouse IgM, igG1, IgG2a, IgG2b, IgG3 and IgA, Bio-Rad} in dilution buffer (PBS / 0.1 % Tween).
Add 100 pL per well into appropriate wells and incubate 60 min at 37° C.
9. Wash as in step 3.
10. Dilute alkaline phosphatase conjugated anti-rabbit IgG (Tago cat # 4620) in PBS / 0.1 % Tween to 1 :2000 concentration. Add 100 pL per well and incubate at 37° C for 60 min.

11 . Wash as in step 3.

12. Prepare enzyme substrate using Sigma #104 alkaline phosphatase substrate tablets (1 mg/mL in 10 % diethanolamine substrate buffer). Add 100 uL per well and incubate in the dark at room temperature. Absorbance can be read at 405 nm at approximately 15-min intervals.

13. Absorbance readings may be converted to pg antibody per mL serum using dose-response curves generated from ELISA responses of the rabbit anti-mouse isotype antibodies to various concentrations of mouse class and subclass specific immunoglobulins (Zymed Labs. Inc.l.
The procedure used to determine antibody binding to specific sites of rapamycin and to quantify antibody cross-reactivity to FK-506, cyclosporine, and KLH or HSA proteins was as follows:
Inhibition ELISA Protocol:
1. Use Falcon Pro-bind immunoplates.
2. Dilute coating antigen to 1 ~ig/mL in carbonate-bicarbonate buffer. Add 100 pL per well and incubate overnight at 4° C.
3. On the same day prepare inhibiting antigen tubes. Aliquot antibodies into glass test tubes. Prepare appropriate antigen concentration in ethanol and add to aliquoted antibody at 10 ~L ethanol solution / 250 pL antibody. Vortex tubes and incubate overnight at 4° C.
4. Shake out wells and wash 3x with 200 ~L PBS / 0.05 % Tween per well.
5. Add 200 pL blocking buffer per well (PBS / 2 % BSA1. Incubate 60 min at room temperature.
6. Wash as in step 4.
7. Transfer contents of inhibition tubes to antigen-coated plate, 100 pL per well.
Incubate 60 min at 37° C.
8. Wash as in step 4.
9. Dilute alkaline phosphatase conjugated anti-mouse IgG (Tago cat # AMI 4405?
in PBS / 0.1 % Tween to 1 :2000 concentration. Add 100 pL per well and incubate at 37° C for 60 min.
10. Wash as in step 4.
11. Prepare enzyme substrate using Sigma #104 alkaline phosphatase substrate tablets (1 mg/mL in 10 % diethanolamine substrate buffer). Add i00 ~L per well and incubate in the dark at room temperature. Absorbance can be read at 405 nm at approximately 15-min intervals.
Buffers used in the direct, isotyping and inhibition ELISA protocols were:
Coatinct buffer (sodium carbonate/bicarbonate 0 05 M pH 9 61 Sodium carbonate (Fisher, cat # S-233-500) 2.93 Sodium bicarbonate (Fisher, cat # S-263-500) 1.59 -adjust pH to 9.6 using 1 M HCI or 1 M NaOH
-store at 4° C
10x PBS buffer Potassium phosphate, mono-basic (Fisher, cat P-2848-500?
8.00 g Sodium phosphate, di-basic (Fisher, c:at # S-373-1 ) 46.00 g Sodium chloride (Fisher, cat # S-671-3) 320.00 g Potassium chloride (Fisher, cat # P-217-500) 8.00 g -dissolve in 4 L distilled water -store at room temperature Dilution buffer (1x PBS 10.1 % Twec:nl -10x PBS 50.0 mL
-distilled water 450 mL
-Tween-20 (Polyoxyethylene-sorbitol monolaurate Sigma, cat # P-1379) 0.5 mL
-adjust pH to 7.2 and store at room temperature Wash buffer 11 x PBS / 0.05 % Tween~
-10x PBS 200 mL
-distilled water 1800 mL
-Tween-20 1 .0 mL
-adjust pH to 7.2 and store at room temperature Blocking buffer (1 x PBS / 2 % BSA) -1 x PBS 100 mL
-Bovine Serum Albumin (Sigma, cat It A-7030) 2.0 g -store at 4° C
Substrate buffer (10 % diethanolamii~
Diethanolamine (Fisher, cat # D-45-500) 97.0 mL
Magnesium chloride (Fisher, cat # M-33-500) 100.0 mg -adjust pH to 9.8 and store at 4° C (protect from light) The direct ELISA, isotyping and inhibition ELISA procedures have been described to detect mouse antibodies (poly- and monoclonal antibodies), however these procedures can be modified for other species, including but not limited to antibodies of rabbit, guinea pig, sheep or goat.
Example 8 - Polyclonal Antibody Responses to the Rapa 42 DVS Immunoaen:
Polyclonal antiserum was prepared in mice, chicken and rabbits using the Rapa-42 immunogen described in Example 1 and the immunization regimes described in Example 6. The ELISA reactivity of rabbit and chicken serum (7 days post-tertiary injection) to Rapa and FK-HSA conjugates is shown in Table 1 .
Table 1 : Rabbit and Chicken Polyclonal Antibody (Rapa-42-DVS-KLH Immunogen) Reactivity to Rapa and FK (O.D. at 405 nm)Rabbit and Chicken Polyclonal Antibody fRapa-DVS-KLH Immunogen) Reactivity to Rapa and FK (O.D. at 405 NM)Rabbit and Chicken Polyclonal Antibody /Rapa-DVS-KLH Immunogen) Reactivity to Rapa and FK (O.D. at 405 NM).
ntigens Rabbit #1 Rabbit #2 Chicken SA-HAS-HAS 1.634 2.528 1.537 0.039 1 .478 0.632 0.128 0.122 0.176 Rabbits # 1 and # 2 showed good antibody reactivity to the Rapa antigen with O.D.'s. at 405 nm of 1.634 and 2.528 respectively. The serum dilution from rabbit # 1 showed low cross-reactivity to the FK antigen (2.3 %) and low non-specific reactivity to the HSA carrier molecule (7.8 %). The serum dilution from rabbit # 2 however, displayed substantial cross-reactivity with the FK antigen (58.5 %), non-specific reactivity to the HSA carrier was low 14.8 %).
The IgY recovered from eggs (PEG isolation method) of a Rapa immunized chicken had good reactivity to the Rapa antigen and showed a 41 % cross-reactivity with the FK antigen. Non-specific reactivity to the HSA carrier was low at 1 1.5 %.
The serum from rabbit # 1, having the best specificity to the Rapa antigen, was used in an inhibition ELISA assay, the results are shown in Table 2.
Table 2: Percent Inhibition of Rabbit and Chicken Polyclonal Antibodies by Rapa, FK, CSA, Rapa and FK Metabolites.
Inhibiting antigens Rabbit #1 Chicken Rapa 48 30 Met 1 18 34 Met 2 22 0 Met 3 15 35 Met 4 28 1 g Met 5 16 35 Met 1 O p Met 2 0 0 Met 3 0 0 Met 4 0 13 Met 5 0 0 This serum was inhibited 48 % by Rapa. Rapa metabolites 1-5 showed marginal inhibition from 15-28 % (metabolite specificities listed in table 3). CSA, FK
or FK
metabolites 1-5 showed no inhibition, the KLH and HSA proteins did not inhibit 5 antibody binding to the Rapa antigen coated ELISA plate. The chicken IgY
prep demonstrated less inhibition with Rapa or i:he 5 Rapa metabolites and no inhibition with FK, CSA, KLH or HSA proteins or 4 of the FK metabolites (FK metabolite #

showed a low level of inhibition).
10 Table 3 : List of Rapamycin and FK Metabolites Used in Inhibition ELISA
Assays Rapa metabolites * Functional group identification M1 Hydroxy Rapamycin (Hydroxylation is between C9 and C23) M2 Hydroxy Rapamycin (Hydroxylation is between C1 and C8 or C32 and C36) M3 7-O-Demethyl Rapamycin M4 41-O-Demethyl Rapamycin M5 32, 41- Didemethyl Rapamycin FK metabolites * *
M 1 13-O-Demethyl M2 15-O-Demethyl M3 31-O-Demethyl M4 13,31-O-Didemethyl M5 15,31-O-Didemethyl ~" Rapa metabolites were isolated by procedures described in example 5.
'* FK metabolites were isolated by procedures known in the art.
Balb/c female mice immunized (1 °, 2°, 3° and 2 booster injections) with the Rapa-DVS-KLH immunogen (as described in Example 1 ) or with the Rapa-suc-KLH
immunogen (as described in Example 2) showed good reactivity to the Rapa antigen (direct ELISA results shown in Table 4), with low non-specific reactivity to the HSA carrier molecule. However, the sera from mice immunized with the Rapa-suc-KLH immunogen showed high cross-reactivity with the FK antigen, displaying 92.5 %, 57.4 % and 60.2 % FK cross-reactivity with mouse # 1, 2 and 3 respectively. With sera from mice immunized with the Rapa-DVS-KLH
immunogen, the FK cross-reactivity was much less, at only 1 1 .6 %, 33.4 % and 6.7 % for mice # 4, 5 and 6 respectively. These results demonstrate that the Rapa-DVS conjugates elicit Rapa-specific antibody, while the Rapa-suc conjugate elicits antibody with marked cross-reactivity to the FK antigen. The DVS
conjugates of this invention are preferred for producing Rapa-specific antibodies.
Table 5 shows the sera reactivity from four Balb/c (Rapa-DVS immunogen, 1 °, 2°, 3° and booster injections) mice used in fusion procedures of the invention. All four mice had good antibody levels (high O.D.'s by direct ELISA to Rapa-HSA) with little or no non-specific reactivity to the carrier protein, HSA. As was shown with the results in Table 4, the cross-reactivity to the FK antigen was very fow, mice 7, 8, 9 and 10 having only 12.4 %, 13.9 %, 15.6 % and 19.9 % FK cross-reactivity respectively. This result again demonstrates the utility of DVS-immunogen for eliciting rapamycin specific antibodies. The Rapa-DVS immunogen elicited high titer antibody to the Rapa antigen. Table 6 shows that the Rapa-DVS mouse # 7 serum had substantial antibody reactivity to the Rapa antigen at a 1 :800 dilution and that mouse # 10 serum had good antibody reactivity to Rapa-antigen at a 1:6400 dilution.
Table 4 : Mouse Polyclonal Antibody (Rapa-suc-KLH or Rapa-DVS-KLH
Immunogens) Reactivity to Rapa and FK (O.D. at 405 nm) ELISA Rapa-sucRaha-sncRaga-sucRala-DVSRapa-DVSRala-DVS

antigens#1 #2 #3 #4 #5 #6 Raga-IiSA-1 .51 1 .8 1 - 18 I .891 1 .808 1 .606 11SA-I-iSA

FK-HSA 1.405 1.0A3 1.072 0.220 0.603 0.108 HSA 0.019 O.OO9 0.005 0.013 0.011 0.016 Table 5 : Mouse Polyclonal Antibody Fleactivity (Rapa-DVS-KLH immunized pre fusion bleeds) to Rapa and FK
Rapa-DVS Rapa-DVS Rapa-DVS Rapa-DVS
#7 #8 #9 #10 ELISA

antigens Rapa-HSA- 2.403 1.279 2.061 1.707 HSA-HSA

FK-HSA 0.298 0.179 0.322 0.340 HSA 0.033 0.006 0.050 0.010 Table 6: Titration of Mouse Polyclonal Sera to Rapamycin (O.D. at 405 nm) Dilution Rapa-DVS # 7 Rapa-DVS # 10 1 :100 3.265 3.120 1:200 3.161 3.216 1 :400 2.201 3.090 1:800 1.369 3.153 1:1600 0.674 2.635 1:3200 0.388 1.872 1 :6400 0.219 1.090 Mouse polyclonal sera to the R,apa-DVS immunogen had little or no cross-reactivity to FK (confirming result in Table 5), CSA, KLH or HSA epitopes as demonstrated by inhibition ELISA results shown in Table 7. These sera showed significant inhibition with the Rapa antigen (approximately 50 %) with varying levels of inhibition with the rapamycin metabolites (M1-M5).
The results shown in Table 8 demonstrate that this inhibition was Rapa concentration dependent. Rapa significantly inhibited antibody binding at 2.5 - 15 0.1 5 Ng concentrations, little inhibition was seen at the 0.04 Mfg Rapa concentration. The Rapa-HSA inhibitor showed a similar dose-dependent inhibition of anti-Rapa antibody binding. No inhibition occurred when HSA or KLH protein SUBSTITUTE SHEET (RULE 26) was used as inhibitor antigens in this assay.
Table 8: Rapa Concentration Dependent Inhibition of Mouse Polyclonal Antibody (Rapa-DVS-KLH immunized pre-fusion bleed) Inhibiting Ag Percent InhibitionPercent Inhibition concentration Rapa Rapa-42-HSA
(pg) 2.5 78 97 1.25 67 94 0.62 57 86 0.31 44 83 0.15 37 74 0.08 21 64 0.04 13 50 * No inhibition with HSA or KLH
Example 9 - A Method for Monoclonal Antibody Production (MoAb):
The steps for monoclonal antibody production are summarized below:
Immunize mice with parent and metabolite conjugates (1, 2, 3 & boost) U
Recover Ab secreting B cells from mouse spleen Myeioma cell lines (NS-1, SP-2, and P3X63-Ag8.653) U
Hybridization (using PEG) U
Propagation 2o a Screening (Immunoblot, ELISA, automated assays) U
Cloning (3x) U
Screening U
Propagation U
Characterization (metabolite cross-reactivity) U
Tissue culture MoAb production U
Ascites MoAb production . 35 The procedure used to produce the monoclonal antibodies of the invention is as follows:
Although there are many suitable reagent suppliers, we have found the following to be most preferred for obtaining a high yield of fusion products, for 'I 9 isolating stable clones and for the production of monoclonal antibodies (MoAb).
Dulbecco's Modified Ea4les Medium: (DNIEM) from JRH BIOSCIENCES, Cat #
56499-10L + 3.7 g/L NaHC03 HAT supplement: (100x - 10 mM sodium hypoxanthine, 40 mM aminopterin, 1.6 mM thymidine) from CANADIAN LIFE TECHNOLOGIES, Cat #

HT stock: (100x - 10 mM sodium hypozanthine, 1.0 mM thymidinelfrom CANADIAN LIFE TECHNOLOGIES, Cat # 1 1067-030 FCS : CPSR-3 Hybrid-MAX from SIGMA, Cat # C-9155 Polvethylene glycol (PEG): Use PEG 4000, SERVA # 33136. Autoclave PEG, cool slightly and dilute to 50 % (w/v) with serum free DMEM. Make fresh PEG the day before the fusion, and place in 37° incubator.
Fusion Procedure:
Myeloma cells should be thawed and expanded one week before fusion and split the day before the fusion. Do not keep the myeloma cell line in continuous culture. This prevents the cells from becoming infected with mycoplasma and also from any changes, which may result from repeated passaging.
For example:
SP2/0 can be split back to 1x104 ceIIsImL, freeze at least 5x106 cells/vial NS-1 can be split back to 1x104cells/mL, freeze at least 5x106 cellslvial P3X63-Ag8.653 can be split back to 1 x104 cells/mL, freeze at least 5x106 cell/vial Culture the myeloma cell line so that you will have at least 0.5x10' cells (in log phase growth) on the day of the fusion. Three to five days prior to fusion, boost the immunized mouse. The mouse must be genotypically compatible with the myeloma cell line. Myeloma cell drug sensitivity should be confirmed.
Serum should be tested for its ability to support growth of the parental myeloma cell line. To test batches of serum, clone the parental myeloma cells (as outlined under cloning) in 10 %, 5 %, 2.Ei %, and 1 % FCS. No feeder layer is required. Check growth and cell viability daily for 5 days.
Fusion Day 1 . Place fresh medium, FCS to be used in fusion in water bath.
2. Harvest myeloma cells and wash 3x with serum-free medium (DMEM, RPMI or other commercially available tissue culture media may be used).

3. Remove spleen (lymph node cells may also be used) from immunized mouse;
resterilize instruments or use new sterile instruments between each step, i.e.
cutting skin, cutting abdominal muscle, removing spleen.
4. Rinse outside of spleen 3x by transferring to plastic petri plates containing 5 sterile medium; use sterile forceps between each step.
5. Place spleen in plastic petri dish with serum-free medium in it, cut into 4 pieces and push gently through screen with sterile glass plunger to obtain a single cell suspension.
6. Centrifuge spleen cells in 50-mL conical centrifuge tubes at 300x g (1200 rpm 10 in silencer) for 10 minutes.
7. Resuspend in 10-mL medium. Dilute an aliquot 100x and count cells.
8. Centrifuge rest of spleen cells, resuspend and recentrifuge. Myeloma cells can be washed at the same time. The NS-1, SP2/0 and P3X63Ag8 myeloma cell lines are most preferred, however other myeloma cell lines known in the art 15 may be utilized. These include, but are not limited to, the mouse cell lines:
X63Ag8.653, FO, NSO/1, FOX-NY; rat cell lines; Y3-Ag1.2.3, YB2/0 and IR983F and various rabbit and human cell lines.
9. Add myeloma and spleen cells together in 5:1 or 10:1 ratio with spleen cells in excess.
20 10. Recentrifuge: spleen cells and myeloma have now been washed 3x.
1 1. Gently flick pellet and place in incubator for 15 minutes to reach 37° C.
Fusion Protocol:
1. Add 1 mL of 50 % PEG solution over 1 minute stirring (add 0.25 mL/15 sec) holding tube in 37° C water bath (beaker with warm water). PEG fuses membranes of myeloma with antibody secreting (B) cells.
2. Stir 1-minute holding in 37° C water bath. Solution will turn lumpy.
3. Add 1-mL medium at 37° C over 1-minute stirring.
4. Add another mL medium over 1-minute stirring.
5. Add 8-mL medium over 2 minutes stirring.
6. Centrifuge for 10 minutes at 300x g (1200 rpm in silencer) and pipet off supernatant.
7. Add 10 mL medium + 20 % FCS (v/v) to cells in tube and pour into plastic petri dish.
8. Leave in incubator with 5 % COZ at 37° C for 1-3 hours. This enhances stability of fusion products.
9. Plate cells out at a concentration of 2x105 cells per wel! in medium f100 pL /
well).

10. Feed cells 100 ~L of 2x HAT in medium the next day.
- No feeder layer is necessary at this time - Feed fusion products 100 pL medium + HAT selection additive on day 3.
Hybridoma cells (myeloma:spleen cell hybrids) are selected by the addition of the drug aminopterin which blocks the de novo synthesis pathway of nucleotides. Myelomaapleen hybrid cells can survive by use of the salvage pathway. Unfused myeloma cells and myeloma:myeloma fusion products have a defect in an enzyme of the salvage pathway and will die.
Unfused spleen cells from the immunized mouse do not grow in tissue culture. Other drugs known in thc: art may be used to select myelomaapleen cell hybrids, such as methotrexate or azaserine.
- Feed fusion products 100 pL medium + HAT + spleen / thymus feeder layer if necessary on day 5 f 1 x10'' cells / well). Fibroblasts, RBC's or other cell types may also be used as feeder layers.
- Continue to feed cells medium + HAT for 1 week, by day 7 post-fusion, change to medium + HT. Clones should appear 10-14 days after fusion.
Note:
1. Washing of the spleen cells, myeloma cells and steps 1-6 of the fusion protocol are performed with serum-free medium.
2. Thymocytes die in about 3 days, non-fused spleen cells in about 6 days.
3. Hybrids are fairly large and almost alv\rays round and iridescent.
4. T-cell and granulocyte colonies may also grow. They are smaller cells.
To Clone Hybrid Cells:
1 . Resuspend the 200 pL in the well with a sterile eppendorf pipet tip and transfer to a small 5-mL sterile tube.
2. Add 200 pL medium (20 % FCS) to the original well. This is a safety precaution of the cloning procedure. Parent cells may also be transferred to 24 well plates as a precaution.
3. Take 20 ~L of the hybrid cell suspension from step 1 and add 20 ~L of eosin or trypan blue solution. Under 40x magnification hybrid cells appear to be approximately the same size and morphology as the myeloma cell line.
4. Clone viable cells by limiting dilution with:
20 % FCS used in fusion medium - 1 x HT
1x106 thymocytes per mL
clone 1400 cells per cloning protocol Dilution Cloning Procedure Make 10 mL of thymocyte cloning suspension in DMEM with 20 % FCS. Take 1400 hybrid cells and dilute to 2.8 mL.
Row 1 : Plate 8 wells (200 pL / well) - 100 cells / well.
To the remaining 1.2-mL add 1 .2-mL medium.
Row 2: Plate 8 wells (200 pL / well) - 50 cells / well.
To the remainder add 2.0-mL medium.
Row 3: Plate 8 wells (200pL / well) ~ 70 cells / well To the remainder add 1 .2-mL medium.
Row 4: Plate 8 wells (200 pL / well) ~ 5 cells / well.
To the remainder add 2.8-mL medium.
Row 5 & 6: Plate 16 wells (200 ~L / well) ~ 1 cell ! well.
After cloning and screening for positive wells, re-clone the faster growing, stronger reacting clones. To ensure that a hybridoma is stable and single-cell cloned, this cloning is repeated 3 times until every well tested is positive.
Cells can then be grown up and the tissue culture supernatants collected for the monoclonal antibody. Other limiting dilution cloning procedures known in the art, single-cell cloning procedures to pick single cells, and single-cell cloning by growth in soft agar may also be employed.
Monoclonal Antibody Production Monoclonal antibodies can be readily recovered from tissue culture supernatants. Hybrid cells can be grown in tissue culture media with FCS
supplements or in serum-free media known in the art. Large-scale amounts of monoclonal antibodies can be produced using hollow fibre or bioreactor technology. The concentration, affinity and avidity of specific monoclonal antibodies can be increased when produced as ascitic fluid.
Ascitic Fluid Production 1. Condition mice by injecting (I.P.) 0.5-mL pristane (2, 6, 10, 14-tetramethylpentadecane) at least 5 days before hybrid cell are injected. Mice should be genotypically compatible with cells injected, i.e., Balb/c mice should be used with fVS-1 or SP2/0 fusion products. Mice of non-compatible genotype may be used if irradiated before cells are injected. However, Balb/c pristane treated mice are the best to use.
2. Inject (I.P.) 106 for more) hybrid cells in PBS. Wash cells 3x prior to injection to remove the FCS.
3. Mice will be ready to tap in about 7-14 days. Use an 18-~/z G needle to 2:3 - harvest ascites cells and fluid.
4. Transfer at least 106 ascites cells from these mice to more pristane treated mice.
5. Ascites cells can be frozen in 10 % DIVISO, 20 % FCS, DMEM medium.
Freeze about 5x106 cells per vial.
Monoclonal antibodies prepared in tissue culture or by ascitic fluid may be purified using methods known in the art.
Example 10 - Isolation and Characterizatie~n of Monoclonal Antibodies to Specific Sites of Rapamycin:
The steps to isolate and characterize monoclonal antibodies with reactivity to a specific sitelsl of rapamycin are outlined below:
Steps to Identify MoAb to Specific Sites of Rapamycin:
Immunization regime (collect polyclonal sera) U
Direct ELISA IAb to Rapa) ll Inhibition ELISA
(specificity to Rapa, cross-reactivity to FK, CSA, KLH or HSA inhibitors) U
Direct ELISA (FK or HSA cross-reactivity) U
Fusion procedure U
Screening of parent fusion products Immunodot Direct ).LISA
Inhibition ELISA
Ab isotyping U
Cloning and screening (3x) ll Characterization of Ab in tissue culture supernatant Direct ELISA ( IgC3 isotypes only) Direct ELISA (FK or HSA cross-reactivity) Inhibition ELISA (Rapa, FK, CSA, Flapa and FK derivative inhibitors) U
Ascites production Direct ELISA (Ab titre and isotype) Inhibition ELISA (Rapa, FK, CSA, Flapa and FK derivative inhibitors) U
Ab purific:ation - characterize antibody reactivity Parent fusion products from myetomaapleen cells of Rapa-42 (Example 1 ) immunized mice were initially screened by an immunodot assay as follows:
Immunodot Assay 1 . Dot 5-10 pL of antibody onto nitrocellulose paper, which has been girdded for reference.
2. Air-dry and immerse nitrocellulose in PBS / 0.1 % Tween / 5 % milk /v/v/w) to block non-specific binding sites. Incubate at room temperature for 60 min with shaking.
3. Rinse twice with PBS / 0.05 % Tween and wash with shaking for 10 min.
4. Dilute alkaline phosphatase conjugated anti-mouse IgG (Tago cat # AMI 4405) in PBS / 0.1 % Tween to 1 :2000. Place nitrocellulose on parafilm or saran wrap and add diluted conjugated antibody until nitrocellulose is covered.
Incubate covered at 37° C for 60 min. Do not allow nitrocellulose to dry out between steps.
5. Wash as in step 3.
6. Prepare enzyme substrate using BCIP / NBT (Canadian Life Technologies, cat #
18280-016; 88 frL NBT and 66 ~L BCIP in 20 mL substrate buffer, 100 mM
Tris, 5 mM MgCl2, 100 mM NaCI). Place nitrocellulose in substrate solution and shake at room temperature for 10 - 30 min, watching for color development.
7. Rinse nitrocellulose with water to stop reaction.
Once antibody secreting parent fusion products were identified, the tissue culture supernatants were further characterized for rapamycin reactivity by the direct, isotyping and inhibition ELISA assays as described in Example 7.
Tissue culture supernatants from clones (3x) of rapamycin positive parent fusion products were then characterized by isotyping ELISA to isolate IgG producing clones, by direct ELISA to determine FK and HSA cross-reactivity and by inhibition ELISA
using Rapa, CSA, FK and Rapa and FK metabolites to determine specificity and rapamycin site reactivity.
Using the immunodot and direct ELISA assays over 600 parent fusion products were identified which have strong reactivity to the Rapa antigen. Of these parent products, over 200 have been cloned, tested for reactivity to Rapa by direct ELISA, and 100 positive clones were then re-cloned (2x). We have now isolated many IgM and IgG secreting clones with reactivity to the Rapa antigen by direct, inhibition and isotyping ELISA assays. Table 9 illustrates examples of ELISA
reactivity using IgG monoclonal antibodies from various clones of three fusion procedures (R-1, R-2 and R-3).
Table 9 : Monoclonal Antibody (Rapa-DVS-KLH immunogen) Reactivity to Rapa, FK
and HSA (O.D. at 405 nm) !gG Producing Rapa-HSA FK-HSA FK Cross HSA

Clone # Reactivity (%!

R-1-1 1 .929 0.8'.99 46.6 0.005 R-1-2 1 .533 0.933 60.9 0.001 R-1-3 1 .581 1 .0'41 65.8 0.002 R-1-4 2.424 0.372 15.3 0.003 R-1-5 0.654 0.1 17 17.9 0.001 R-2-1 0.881 0.166 18.8 0.003 R-2-2 3.122 0.037 1 .2 0.036 R-2-3 0.655 0.609 92.9 0.033 R-2-4 0.535 0.298 55.7 0.027 R-2-5 1 .603 1 .151 71 .8 0.001 R-2-6 0.440 0.1 16 26.4 0.001 R-3-1 2.825 0.002 0.1 0.005 R-3-2 0.579 0.003 0.5 0.002 The monoclonal antibody reactivity to the Rapa-42 antigen varies from 0.440 to 3.122 O.D. units in these 13 examples. Non-specific reactivity to the carrier HSA
protein is negligible. Monoclonal antibody cross-reactivity to the FK antigen of 5 these clones varies considerably. The clones R-1-4, R-1-5, R-2-1, R-2-2, R-2-6, R-3-1 and R-3-2 show little or only marginal binding to the FK antigen; clones R-and R-2-4 have approximately 50 % cross-reactivity to the FK antigen; clones R-2, R-1-3 and R-2-4 show significant cross-reactivity to FK and clone R-2-3 demonstrates almost equivalent affinity and reactivity for the FK and Rapa 10 antigens. For development of therapeutic drug monitoring assays (TDM) with specificity for Rapa, the clones secreting antibodies with low or little cross-reactivity to the FK antigen would be preferred. Most preferred would be clones which secrete high levels of anti-Rapa IgG monoclonal antibodies with a low level of anti-FK cross-reactivity. Examples of such clones listed in Table 9 would be R-15 1-4, R-2-2 and R-3-1.
Example 11 - Mapping of Rapa Antibody E3indina region To further characterize the reactivity of monoclonal antibodies trom various clones, inhibition ELISA were performed. Table 10 shows example inhibitions 20 from monoclonal antibodies of 4 clones. -fhe monoclonal antibody from the R-clone is significantly inhibited by Rapa (85 %) and by Rapa metabolite # 2 (77 %).
The antibody binding is not inhibited by R,apa metabolite # 1 f 15 %) and moderately inhibited by Rapa metabolites 3, 4 and 5 (32 %, 30 % and 29 respectively). The mapping of reactivity of this monoclonal antibody with the metabolite M1-5 inhibitors, would indicate that the specitic site for antibody binding is between C9 and C23. Hydroxylation in metabolite 1 between C9 and C23 changed a significant epitope in this region necessary for antibody binding.
The low level of inhibition seen with Rapa metabolites 3-5 is most probably due to conformational changes of the parent rapamycin molecule with demethylation of residues 7, 32 and 41, which moderately affect antibody binding in the C9-C23 region. This monoclonal antibody was found to be 46 % cross-reactive with the FK antigen by direct ELISA. The binding of R-1-1 MoAb was also found to be cross-reactive with FK by inhibition ELISA, FK inhibited antibody binding by 43 %.
FK metabolites also significantly inhibited 43-57 %. CSA, KLH or HSA proteins showed no inhibition.
With the R-1-5 MoAb, Rapa and Rapa metabolite # 2 significantly inhibited antibody binding. There was no inhibition with the Rapa metabolite # 1, again suggesting that the specific site of this anti-Rapa antibody is located between C9 and C23 residues. The inhibition noted with Rapa 3-5 metabolites is again believed to be due to conformational changes caused by demethylation of residues 7, 32 and 41, affecting antibody site binding. This monoclonal showed some cross-reactivity with the FK antigen, this cross-reactivity was also observed with all FK metabolites. Cross-reactivity to FK antigen as measured by direct ELISA
was only marginal (Table 9). The R-1-5 MoAb did not bind to CSA, KLH or HSA
proteins.
Chemical derivatization of specific sites on the rapamycin molecule including the carbon residues, nitrogen residues, oxygen, hydroxyl, methoxy or methyl groups will produce compounds similarly useful as the above metabolites for mapping the antibody binding region.
Table 10 : Percent Inhibition of MoAb Tissue Culture Supernatants by Rapa, FK, CSA and Rapa and FK Metabolites Inhibiting R-1-1 R-1-5 R-2-2 R-3-1 antigen Rapa 85 77 83 gg Rapa met 1 15 12 16 3g_2 Rapa met 2 77 91 88 95 Rapa met 3 32 56 26 92 Rapa met 4 30 57 71 96 Rapa met 5 29 44 10 94 FK met 46 2.4 19 0 FK met 57 3~6 16 0 FK met 45 2~6 9 0 FK met 53 3.B 13 0 FK met 46 26 13 0 Rapa and Rapa metabolite # 2 inhibited R-2-2 MoAb binding to Rapa antigen coated ELISA plates. Rapa metabolites 1, 3 and 5 did not significantly inhibit binding, however metabolite # 4 showed :significant inhibition at 71 %. We 5 believe that this might indicate that the MoAb's binding site is again in the C9 to C23 region, that a modification of this region affects binding, as observed with metabolite # 1 and that demethylation at site 41, also affects antibody binding due to conformational changes within the antibody site. The fact that metabolites and 5 have less inhibitory effect than with MoAbs R-1-1 and R-1-5, may be due to a greater affinity of R-2-2 for the antibody binding site (specific antibody epitope) or possibly that R-2-2 MoAb recognizes a sightly different antibody binding epitope in the C9-C23 region than the R-1-1 or R-1-5 MoAbs. Indeed, tissue culture supernatants of R-2-2 showed the highest O.D. reactivity with the Rapa antigen by direct ELISA (Table 9) indicating good antibody affinity / avidity. The fact that R-2-2 MoAb showed very little cross-reactivity with FK or FK metabolites 1-5 again indicates good affinity / avidity with the specific antibody binding site on rapamycin. Even though the FK and raparnycin molecules are structurally similar at the nitrogen ring region (chemical strucl:urel, studies indicate there are conformational differences between these molecules. Three dimensional structure plays an important role in epitope presentation and recognition by the immune system, therefore a MoAb with high affinity, avidity and specificity for a specific three dimensional epitope site of rapamycin, would not necessarily cross-react with molecules of similar chemical structure, such as FK. R-2-2 did not react with CSA, KLH or HSA.
With the R-3-1 MoAb, Rapa and Rapa metabolites 2-5 significantly inhibited antibody binding to sites on the Rapa molE;cule. Rapa metabolite # 1 marginally inhibited antibody binding (38 %) and FK and FK metabolites 1-5, CSA, KLH or HSA showed no inhibition to this MoAb's apecific site on Rapa. Again these results could suggest that the specific antibody binding epitope may be in the C23 region, however unlike previous monoclonal antibody epitope mapping results, the demethylated metabolites showed no inhibiting capacity (i.e. inhibited similar to the parent Rapa molecule). We believe that R-3-1 may recognize the C9-C23 region, or alternately recognize an epitope in the opposite face of the molecule, for example between C24-C36. Identification of the specific site of R-3-1 on the Rapa molecule can be done using various other minor metabolite peaks isolated as described in Example 5.
A further clue that R-3-1 may recognize a different binding site than R-1-1, R-1-5 or R-2-2 was elucidated from results of experiments using various dilution buffers in our inhibition assay. We observed that rapamycin which had been diluted in only aqueous buffer did not inhibit the binding of MoAbs R-1-1, R-1-5 or R-2-2, while rapamycin diluted in aqueous buffer containing 10 % FCS did inhibit binding, possibly indicating that a modification to rapamycin, such as hydrolysis in aqueous buffer, modifies the antibody binding site and no longer binds the MoAbs.
Rapamycin maintained in a buffer less likely to cause hydrolysis (i.e.aqueous buffer containing 10 % FCS), would maintain antibody binding epitope integrity and bind MoAbs R-1-1, R-1-5 or R-2-2. MoAb R-3-1 was inhibited by rapamycin diluted in either aqueous buffer or aqueous buffer containing 10 % FCS. This finding indicates that MoAb R-3-1 recognizes a specific site of rapamycin which is not affected by hydrolysis, a site different from the hydrolysis-sensitive binding site of MoAbs R-1-1, R-1-5 and R-2-2.
Example 12 - Mouse Polyclonal Antibody Responses to the Rapa 27 ox DVS
Immunogen:
Serum from Balb/c mice post-tertiary immunization with the Rapa-27-ox-DVS-KLH immunogen (as in Example 3) was tested for ELISA reactivity to Rapa-42-HSA, Rapa-27-HSA and FK-HSA (Table 1 1 ). These results indicate that serum from mice immunized with the Rapa-ox conjugate may recognize a different epitope on the parent rapamycin molecule than serum from mice immunized with the Rapa-42 conjugate. Anti-Rapa-27 serum reacts strongly with Rapa-27-HSA, but generally shows decreased and variable cross-reactivity with Rapa-42-HSA
(12-63 %). As was observed with the Rapa-42-DVS immunogen, the Rapa-27-ox-DVS immunogen elicited site-specific antibodies to rapamycin which did not cross-react to FK antigens (Table 1 1 ).
Inhibition ELISA data (Table 12) demonstrated that the parent Rapa molecule blocked anti-Rapa-27 antibody binding to Rapa-27-HSA (93 %). CSA, KLH and HSA showed no inhibition. Rapa metabolite # 1 showed significant inhibition at %, indicating that the C9-C23 region of the molecule was not involved with antibody recognition. Hydroxylation in the region between C1-C8 or C32-C36 (metabolite # 2) caused significant loss of inhibiting activity (inhibition only 36 %1, indicating that this region may play a role in antibody recognition. The inhibition observed with the parent molecule was decreased with demethylation at residues 7 and 41 (metabolites # 3 and # 4) from 93 % to 42 % and 37 % respectively.
Rapa metabolite # 5 (demethylated at re:;idues 32 and 41 ) completely abrogated antibody binding to the parent molecule. This demonstrates that demethylation of the 32, 41 sites completely inhibit antibody binding to the epitope recognition site.
However, as demethylation at the 41 si~.e (metabolite # 4) or hydroxylation between the C1-C8 or C32-C36 did not completely inhibit antibody binding, we postulate that the methyl group at the 3 2 site plays the significant role in maintaining the three dimensional structure of the epitope recognition site.
The reduction in inhibiting capacity seen with metabolites 3 and 4 may be due to conformational changes within the antibody epitopes of the C32 region.
Table 11 : Mouse Polyclonal Antibody Reactivity (Raps-27-ox-DVS-KLH
immunogen) to Rapa and FK (O.D. at 405 nm) ELISA Rapa- Rapa- Rapa- Rapa- Rapa- Rapa- Rapa- Rapa-antigen27 27 #12 27 #13 27 #14 27 #15 27 #16 27 27 #11 #17 #18 Rapa- 2.512 2.733 1.592 2.002 1.891 1.769 1.287 0.813 Rapa- 1.594 0.490 0.605 0.968 0.235 0.817 1.538 0.398 FK-HSA0.332 0.091 0.092 0.'094 0.011 0.092 0.070 0.045 HSA 0.257 0.071 0.066 0.061 0.03 0.029 0.002 0.004 Table 12 : Percent Inhibition of Mouse Polyclonal Antibody (Rapa-27-ox-DVS-KLH
immunogen) by Rapa, FK, CSA and Rapa Metabolites Inhibiting Rapa-27 antigens # 12 Rapa 93 Met 1 6g Met 2 38 - Met 3 43 Met 4 37 Met 5 0 Utilizing the Rapa-42-DVS conjugate (Example i ) of the invention to elicit poly or monoclonal antibodies to the C9-C23 region of rapamycin, and the Rapa-27-oxime-DVS conjugate (Example 3) or Rapa-31-DVS conjugate (Example 41 to elicit 5 poly or monoclonal antibodies to other regions of the rapamycin parent molecule, an immunoassay to measure rapamycin and / or rapamycin metabolites is developed. Most preferred would be a TDM assay to specifically measure biologically active rapamycin molecules. Poly and monoclonal antibodies with reactivity to various specific sites of rapamycin can be elicited with the conjugates 10 of the invention.
Example 13 - Measuring the Bioloctical Activity of Rapamycin and Rapamycin Metabolites by in vitro Mixed Lymphocyte Reaction (MLR1 Assay:
The MLR assay is useful for identifying rapamycin metabolites with biological 15 (immunosupressive) activity and to quantify this activity relative to the immunosuppressive activity of the parent rapamycin molecule.
An example of a lymphocyte proliferation assay procedure useful for this purpose is as follows:
1 . Collect blood from two individuals (20 mL each) and isolate lymphocytes using 20 Ficoll-Paque (Pharmacia Biotech).
2. Count lymphocytes at 1:10 dilution in 2 % acetic acid.
3. Prepare 10 mL of each lymphocyte populations (A + B) at 1x106 cells/mL in DMEM / 20 % FCS.
4. Set up a 96 well sterile tissue culture plate, flat bottom (Sarstedt, cat #
25 83.1835). To each well add:
5. Aliquot 100 pL per well lymphocyte population A
6. Aliquot 100 pL per well lymphocyte population B
7. Aliquot 20 pL per well of drug (rapamycin and rapamycin metabolites M1-5) at 0, 2.5, 5, 10, 25, 50 and 100 pglL in triplicate in DMEM with no 30 supplements.
8. To measure the effect of drug on proliferation, incubate the plate for 5 days at 37° C in 5 % CO2 atmosphere.
9. On day 6, prepare 3.2 mL of 1 :50 dilution of Methyl-3H-Thymidine (Amersham Life Science, cat # TRK 120) in DMEM with no supplements. Add 30 yL per WO 98!45333 PCT/CA98/00361 well and incubate for 18 hours at 37° C in 5 % COZ atmosphere.
10. On day 7 cells are harvested onto glass microfiber filters GF/A (Whatman, cat 1820024) using a Cell-Harvestor (Millipore, cat # XX2702550). Wash cells 3x with 1 .0 mL sterile distilled water.
Note: All procedures are done using sterihe techniques in a biological flow hood.
1 1 . Place filters in Scintilation vials and acid 1.5 mL of SciniSafe Plus 50 scintilation fluid (Fisher, cat # SX-25-5).
12. Measure the amount of radioactivity incorporated in the lymphocytes using a beta counter (Micromedic System Inc., TAURUS Automatic Liquid Scintilation Counter) for 1 .0 minute.
13. Calculate averages and standard deviations for each drug and express results as:
Inhibition = [1- Ave CPM of test drug ) x 100 Ave CPM of zero drug Proliferation = 100 - % Inhibition The MLR assay can be utilized to select antibodies of the invention which bind biologically active Rapa metabolites and the parent Rapa molecule. Antibodies could also be selected for reactivity to biologically inactive metabolites.
Example 14 - Immunoassay Kits Usinc~ Polyclonal and Monoclonal Antibodies to Specific Sites of Rapamycin:
The polyclonal and monoclonal antibodies to specific sites of rapamycin of the invention may be used for development of immunoassays or TDM kits. Such assays could include, but are not limited to, direct, inhibition, competitive or sandwich immunoassays (ELISA or other assay systems), RIA, solid or liquid phase assays or automated assay systems.
As will be seen from the foregoing Ex<~mples, procedures not described in detail are conventional. Variations and modifications will be apparent to those skilled in the art and are intended to be encompassed by the above descriptions and the claims appended hereto.
References:
1. Sehgal, S. N., H. Baker & C. Vezina. Rapamycin (AY-22,989), a new antifungal antibiotic: II. Fermentation, isolation and characterization. J.
Antibiot.
(Tokyo) 28:727-732.

2. Streit F., Christians U., Schiebel H.M., Meyer A., Sewing K.F. Structural Identification of Three Metabolites and a Degradation Product of the Macrolide Immunosuppressant Sirolimus (Rapamycin) by Electrospray-MS/MS after Incubation with Human Liver Microsomes. Drug Metab Dispos. 24: 1272, 1996.
3. Wang P.C., Lim H.K., Chan K.W. Isoaltion of Ten Metabolites from the Bile of Rats Receiving Rapamycin (Sirolimus) Intravenously. ISSX Proc. 8: 136, 1995.
4. Nickmilder M.J.M., Latinne D., Verbeeck R.K., Janssens W., Svoboda D., Lhoest G.J.J. Isolation and Identification of New Rapamycin Dihydrdiol Metabolites from Dexamethasone Induced Rat Liver Microsomes. Xenobiotica.
27: 869, 1997.
5. Streit F., Christians U., Schiebel H.M., Napoli K.L., Ernst L., Linck A., Kahan B.D., Sewing K.F. Sensitive and Specific Quantitation of Sirolimus (Rapamycin) and its Metabolites in Blood of Kidney Graft Recipients by HPLC/Electrospray-Mass Spectrometry. Clin Chem. 42: 1417, 1996.
6. Leung L.Y., Zimmerman J., Lim H.K., DiCioccio T.A., Warner L., Nicks D., Chan K., Kantrowitz J, Scatina J, Sisenwine SF, Tonelli AP. Metabolic Disposition of [14C]-Rapamycin (Sirolimus) in Healthy Male Subjects After Single Oral Dose.
ISSX Proc. 12: 26, 1997.

Claims (11)

What is claimed is:

1. A method for producing an antibody which is capable of recognizing a specific region of rapamycin, comprising:
(a) administering an immunogen comprising rapamycin, a linker arm molecule and a protein carrier, to an animal to effect a specific immunogenic response to the rapamycin, (b) recovering an antibody from the animal, and (c) identifying the antibody binding site by measuring the reactivity of the antibody to at least one rapamycin derivative.

2. The method according to claim 1 wherein the step of recovering an antibody comprises recovering at least one antibody producing cell from the animal, immortalizing the antibody producing cell, and isolating a monoclonal antibody from the immortalized antibody producing cell.

3. The method according to claim 1 or 2 further comprising comparing the reactivity of the antibody to at least one rapamycin derivative to the reactivity of the antibody to rapamycin.

4. The method according to any preceding claim wherein the rapamycin derivative is substituted between the C-9 and C-23 position, the C-1 and C-8 position or the C-23 and C-36 position or is demethylated at the 7, 32 or 41 position.

5. The method according to any preceding claim wherein the rapamycin is a rapamycin metabolite.

6. The method according to any preceding claim wherein the linker arm molecule is divinyl sulfone.

7. The method according to any preceding claim wherein the protein carrier is keyhole limpet hemocyanan.

8. The method according to any preceding claim wherein the rapamycin is linked at the 27, 31, 41 or 42 position.

9. The method according to any preceding claim wherein the animal is a member selected from the group consisting of a mouse, rat, rabbit, chicken, guinea pig, goat and sheep.

10. An immunoasay for measuring rapamycin, rapamycin metabolite levels in a mammal comprising:
incubating whole blood, extracted whole blood, sera, extracted sera, urine or feces from the mammal with an antibody produced by a method as claimed in any preceding claim and measuring the quantity of rapamycin, rapamycin metabolite bound to the antibody.

11. An immunoassay kit comprising an antibody produced by a method as claimed in any of claims 1 to 9.