WO1998045333A1 - Method for production of antibodies to specific sites of rapamycin - Google Patents
Method for production of antibodies to specific sites of rapamycin Download PDFInfo
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- WO1998045333A1 WO1998045333A1 PCT/CA1998/000361 CA9800361W WO9845333A1 WO 1998045333 A1 WO1998045333 A1 WO 1998045333A1 CA 9800361 W CA9800361 W CA 9800361W WO 9845333 A1 WO9845333 A1 WO 9845333A1
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- rapamycin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/14—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
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- This invention relates to the production of polyclonal and monoclonal antibodies to specific sites of rapamycin (Sirolimus).
- rapamycin rapamycin
- the reactivity of these poly and monoclonal antibodies make them particularly useful for immunoassays for therapeutic drug monitoring (TDM).
- TDM therapeutic drug monitoring
- 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 (Rapa) is a macrocyclic antibiotic, which was originally isolated in soil samples from Easter Island from a Streptomyces hygroscopicus strain. 1 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:
- CSA cyclosporine
- FK FK- 506
- Therapeutic monitoring of concentrations of these drugs in blood is required to optimize dosing regimes to ensure maximal immunosuppression with minimal toxicity.
- 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 P 450 3A4 enzyme metabolizes rapamycin to a number of demethylated and hydroxylated 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. at. structurally identified four rapamycin metabolites from rabbit liver microsomes. 2 These include 41 -demethyl 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 1 6 hydroxylated and or demethylated metabolites in the bile of rapamycin treated rats. 3 Nickmilder et. at.
- rapamycin represented approximately 35 % of the total radioactivity in blood and that 41 - demethyl, 7-demethyl, and several hydroxy, hydroxydemethyl, and didemethyi 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.
- 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. The following examples describe the best mode for carrying out the invention.
- VPS divinyl sulfone
- Example 1 Synthesis of Rapamvcin-42-Divinyl Sulfone and Conjugation to a Protein Carrier Preparation of rapamycin-42 divinyl sulfone hapten: Rapamycin (0.5 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.
- 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.
- 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 dichloromethane 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.
- 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 7.6) . 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.
- KLH keyhole limpet hemocyanin
- HSA human serum albumin
- rapamycin-42-O-hemisuccinate Dimethylaminopyridine ( 1 1 .8 mg, 97 ⁇ mol) was added to a solution of rapamycin (80.0 mg, 88 ⁇ mol) and succinic anhydride (30.7 mg, 307 ⁇ mol) 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.
- rapamycin-42-O-hemisuccinate (1013.5 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 42- O-succinimidooxysuccinyl rapamycin (1 1 10.5 daltons) was identified as the sodium adduct ( 1 133.5 daltons) by electrospray ionization mass spectrometry.
- 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.
- KLH keyhole limpet hemocyanin
- HSA human serum albumin
- Example 3 Synthesis of Rapamvcin-27-Oxime-Divinyl Sulfone and Conjugation to a Protein Carrier: Preparation of rapamycin-27-oxime: Hydroxylamine hydrochloride (3.0 mg, 44 ⁇ mol) in 100 mL of water was added to a solution of rapamycin (20.0 mg, 22 ⁇ mol) 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 water, 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.
- rapamycin-27-oxime-divinyl Analysis of rapamycin-27-oxime-divinyl: LC/MS analysis (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) 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.
- 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.
- rapamycin-27-oxime-divinyl-sulfone hapten (Rapa-Ox-DVS): 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 50/50 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.
- Rapa-Ox-DVS species X
- Rapa-Ox-DVS species 2
- Rapa-Ox-DVS (species 3)] which were identified by electrospray ionization mass spectrometry.
- Rapa-Ox-DVS 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 3) .
- the positive-ion fragmentation pattern for Rapa-Ox-DVS (species 3) was again consistent with rapamycin modification through the C-27 position.
- Rapa-Ox-DVS-(X) 2.4 mg ( 10%) Rapa-Ox-DVS-(2) : 3.4 mg ( 1 5 %) Rapa-0x-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) ( 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.
- 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.
- rapamycin-31 -divinyl sulfone hapten (Rapa-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 1 8 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 5 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 40/1 0/50 water/acetonitrile/methanol (containing 1 0% 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%)
- Rapa-31 -DVS-KLH and HSA conjugates were prepared as described in example 3. 5
- a fresh or frozen rabbit liver (not induced) is washed with approximately 750 5 mL of 1 .1 5 % KCI (w/v) and cut into small pieces (approximately 5 mm 3 ) . These are placed into a small conical 50 mL centrifuge tube with 1 5 mL of 1 .1 5 % 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 1 0,000 xg for 20 min. Following centrifugation the 0 supernatant is decanted into specialized centrifuge tubes and placed on ice.
- microsomal pellet which contains the cytochrome P 450 enzymes required for the metabolism of rapamycin.
- the microsomes are then re-suspended in 1 .1 5 % KCI, tested for protein concentration using the Lowry method, and 5 stored at -70°C.
- 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 M sodium phosphate buffer (pH 7.4), 0.5 mM EDTA, 5.0 mM MgCI 2 , 3.5 mM
- NADPH 1 .5 mM NADP, 50 mM glucose-6-phosphate, 10 units per mL of glucose-
- 6-phosphate dehydrogenase 6-phosphate dehydrogenase, and 1 0 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 mm 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.
- the metabolites are isolated by thawing the stored reaction mixtures and transferring them to 500 mL glass bottles (100 mL of reaction mixture per bottle)
- This solution is acidified with an equal volume of 0.2 M acetic acid (pH 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.
- a Waters chromatographic system comprised of a 600E gradient controller plus pump, 71 7 autosampler, 486 UV 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:
- peaks were collected, pooled, and labeled. Each of these peaks represents a rapamycin metabol ⁇ te(s) Using the same chromatograhic system, the peaks collected are subjected to further purification using a Waters C1 8 reverse phase (3.6 x 1 50 mm) Symmetry column. The column temperature utilized was 60°C, the flow was 1 .0 mL/min, and the mobile phase consisted of a water/ methanol gradient that was specific for each metabolite purified.
- mice are immunized on day 0 ( 1 ° - primary immunization), day 7 (2° - secondary immunization) , and day 28 (3° - tertiary immunization) by subcutaneous or i ntrape ⁇ toneal injection with rapamycin - conjugate immunogens at doses of 5, 1 0, 1 5, or 20 ⁇ g based on protein content.
- Mice were bled 7-10 days post 2° and 3° i mmun i zation to collect serum to assay antibody responses.
- i mmun i zat i on 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°) and day 60 (3°).
- 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 (or lymph node t i ssues) . This final boost is administered 2 to 3 weeks after the previous in j ection to allow circulating antibody levels to drop off.
- Such i mmunization schedules are useful to immunize mice with rapamycin 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 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, 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 (CytRx), 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 polyclonal antibody, and to stimulate
- B-cells for specific monoclonal antibody production 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.
- the basic direct ELISA protocol for determining antibody reactivity to rapamycin used in the invention was as follows:
- Isotyping ELISA Protocol 1 . Use Falcon Pro-bind immunoplates.
- step 3 Wash as in step 3. 8. Prepare 1 :2 dilution of EIA grade mouse type (rabbit anti-mouse IgM, lgG 1 , lgG2a, lgG2b, lgG3 and IgA, Bio-Rad) in dilution buffer (PBS / 0.1 % Tween) . Add 100 ⁇ L per well into appropriate wells and incubate 60 min at 37° C.
- EIA grade mouse type rabbit anti-mouse IgM, lgG 1 , lgG2a, lgG2b, lgG3 and IgA, Bio-Rad
- Absorbance readings may be converted to ⁇ g 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.) .
- 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:
- Blocking buffer 1 x PBS / 2 % BSA
- Example 8 Polyclonal Antibody Responses to the Rapa-42-DVS Immunogen: Polyclonal antiserum was prepared in mice, chicken and rabbits using the
- 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 (Rapa-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).
- 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 %), nonspecific reactivity to the HSA carrier was low (4.8 %) .
- 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.
- Rapa metabolites 1 -5 showed marginal inhibition from 1 5-28 % (metabolite specificities listed in table 3) .
- CSA, FK or FK metabolites 1 -5 showed no inhibition, the KLH and HSA proteins did not inhibit antibody binding to the Rapa antigen coated ELISA plate.
- the chicken IgY prep demonstrated less inhibition with Rapa or the 5 Rapa metabolites and no inhibition with FK, CSA, KLH or HSA proteins or 4 of the FK metabolites (FK metabolite # 4 showed a low level of inhibition) .
- Rapa metabolites were isolated by procedures described in example 5. * * FK metabolites were isolated by procedures known in the art.
- 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.
- 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.
- 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 low, mice 7, 8, 9 and 10 having only 1 2.4 %, 1 3.9 %, 1 5.6 % and 1 9.9 % FK cross-reactivity respectively. This result again demonstrates the utility of DVS-immunogen for eliciting rapamycin specific antibodies.
- 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 sue Rapa sue Rapa sue Rapa U b napa-uva rtajj v antigens #1 #3 #4 #5
- Example 9 A Method for Monoclonal Antibody Production (MoAb): The steps for monoclonal antibody production are summarized below:
- Dulbecco's Modified Eagles Medium (DMEM) from JRH BIOSCIENCES, Cat # 56499-1 OL + 3.7 g/L NaHC03 HAT supplement: (100x - 10 mM sodium hypoxanthine, 40 mM aminopterin, 1 .6 mM thymidine) from CANADIAN LIFE TECHNOLOGIES, Cat # 31062-037 HT stock: (100x - 10 mM sodium hypozanthine, 1 .0 mM thymidine)from CANADIAN LIFE TECHNOLOGIES, Cat # 1 1067-030 FCS : CPSR-3 Hybrid-MAX from SIGMA, Cat # C-91 55
- 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.
- SP2/0 can be split back to 1 x10 4 cells/mL, freeze at least 5x10 6 cells/vial
- NS-1 can be split back to 1 x10 4 cells/mL, freeze at least 5x10 6 cells/vial
- P3X63-Ag8.653 can be split back to 1 x10 4 cells/mL, freeze at least 5x10 6 cell/vial
- the myeloma cell line so that you will have at least 0.5x10 7 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 1 0 %, 5 %, 2.5 %, and 1 % FCS. No feeder layer is required. Check growth and cell viability daily for 5 days.
- FCS Place fresh medium, FCS to be used in fusion in water bath.
- NS-1 , SP2/0 and P3X63Ag8 myeloma cell lines are most preferred, however other myeloma cell lines known in the art may be utilized. These include, but are not limited to, the mouse cell lines:
- Hybridoma cells (myeloma:spleen cell hybrids) are selected by the addition of the drug aminopterin which blocks the de novo synthesis pathway of nucleotides. Myeloma:spleen 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 the art may be used to select mye!oma:spleen cell hybrids, such as methotrexate or azaserine.
- Feed fusion products 100 ⁇ L medium + HAT + spleen / thymus feeder layer if necessary on day 5 (1 x10 5 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:
- washing of the spleen cells, myeloma cells and steps 1 -6 of the fusion protocol are performed with serum-free medium.
- Thymocytes die in about 3 days, non-fused spleen cells in about 6 days.
- Hybrids are fairly large and almost always round and iridescent.
- T-cell and granulocyte colonies may also grow. They are smaller cells.
- Row 4 Plate 8 wells (200 ⁇ L / well) - 5 cells / well.
- 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.
- 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 NS-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.
- mice will be ready to tap in about 7-14 days. Use an 18-V2 G needle to harvest ascites cells and fluid.
- Ascites cells can be frozen in 10 % DMSO, 20 % FCS, DMEM medium. Freeze about 5x10 6 cells per vial.
- Monoclonal antibodies prepared in tissue culture or by ascitic fluid may be purified using methods known in the art.
- mice Parent fusion products from myeloma:spleen cells of Rapa-42 (Example 1 ) immunized mice were initially screened by an immunodot assay as follows:
- Immunodot Assay 1 Dot 5-10 ⁇ L 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.
- 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.
- 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-1 -1 and R-2-4 have approximately 50 % cross-reactivity to the FK antigen; clones R-1 - 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 0 antigens.
- TDM therapeutic drug monitoring assays
- the clones secreting antibodies with low or little cross- reactivity to the FK antigen would be preferred.
- 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.
- 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 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.
- metabolites 3 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- Rapa metabolite # 1 showed significant inhibition at 69 %, 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 %), 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 residues 32 and 41 ) completely abrogated antibody binding to the parent molecule.
- Table 1 1 Mouse Polyclonal Antibody Reactivity (Rapa-27-ox-DVS-KLH immunogen) to Rapa and FK (O.D. at 405 nm)
- Rapa- 1 .594 0.490 0.605 0.968 0.235 0.81 7 1 .538 0.398
- Table 1 2 Percent Inhibition of Mouse Polyclonal Antibody (Rapa-27-ox-DVS-KLH immunogen) by Rapa, FK, CSA and Rapa Metabolites
- Rapa-42-DVS conjugate (Example 1 ) of the invention Utilizing the Rapa-42-DVS conjugate (Example 1 ) 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 4) to elicit 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 of the invention.
- the MLR assay is useful for identifying rapamycin metabolites with biological (immunosupressive) activity and to quantify this activity relative to the immunosuppressive activity of the parent rapamycin molecule.
- lymphocyte proliferation assay procedure useful for this purpose is as follows:
- 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 Using 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.
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Abstract
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Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
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EP98916710A EP0973805A1 (en) | 1997-04-09 | 1998-04-09 | Method for production of antibodies to specific sites of rapamycin |
AU70207/98A AU740304B2 (en) | 1997-04-09 | 1998-04-09 | Method for production of antibodies to specific sites of rapamycin |
CA002286311A CA2286311A1 (en) | 1997-04-09 | 1998-04-09 | Method for production of antibodies to specific sites of rapamycin |
JP54219898A JP2001518784A (en) | 1997-04-09 | 1998-04-09 | Method for producing antibody against specific site of rapamycin |
Applications Claiming Priority (2)
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US4321597P | 1997-04-09 | 1997-04-09 | |
US60/043,215 | 1997-04-09 |
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US10130998A A-371-Of-International | 1997-04-09 | 1998-07-07 | |
US32599499A Continuation | 1997-04-09 | 1999-06-04 |
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WO1998045333A1 true WO1998045333A1 (en) | 1998-10-15 |
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PCT/CA1998/000361 WO1998045333A1 (en) | 1997-04-09 | 1998-04-09 | Method for production of antibodies to specific sites of rapamycin |
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EP (1) | EP0973805A1 (en) |
JP (1) | JP2001518784A (en) |
AU (1) | AU740304B2 (en) |
CA (1) | CA2286311A1 (en) |
WO (1) | WO1998045333A1 (en) |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000022000A1 (en) * | 1998-10-09 | 2000-04-20 | Isotechnika, Inc. | Methods for the production of antibodies to specific regions of cyclosporine and cyclosporine metabolites |
US6686454B1 (en) | 1998-10-09 | 2004-02-03 | Isotechnika, Inc. | Antibodies to specific regions of cyclosporine related compounds |
WO2006116727A2 (en) | 2005-04-27 | 2006-11-02 | Dade Rehring Inc. | Compositions and methods for detection of sirolimus |
US7445916B2 (en) | 2004-04-14 | 2008-11-04 | Wyeth | Process for preparing rapamycin 42-esters and FK-506 32-esters with dicarboxylic acid, precursors for rapamycin conjugates and antibodies |
US7883855B2 (en) | 2006-07-21 | 2011-02-08 | Abbott Laboratories | Immunosuppressant drug extraction reagent for immunoassays |
US7914999B2 (en) | 2006-12-29 | 2011-03-29 | Abbott Laboratories | Non-denaturing lysis reagent |
US7993851B2 (en) | 2006-12-29 | 2011-08-09 | Abbott Laboratories | Lysis reagent for use with capture-in-solution immunoassay |
US8129127B2 (en) | 2006-12-29 | 2012-03-06 | Abbott Laboratories | Assay for immunosuppressant drugs |
US8221986B2 (en) | 2006-12-29 | 2012-07-17 | Abbott Laboratories | Diagnostic test for the detection of a molecule or drug in whole blood |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2277898A3 (en) * | 2002-07-16 | 2011-06-01 | Biotica Technology Limited | Rapamycin analogues |
EP3368900B1 (en) | 2015-10-29 | 2020-12-23 | Siemens Healthcare Diagnostics Inc. | Sandwich assay for small molecules |
Citations (3)
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WO1993025533A1 (en) * | 1992-06-05 | 1993-12-23 | Abbott Laboratories | Methods and reagents for the determination of immunosuppressive agents |
WO1994024304A1 (en) * | 1993-04-08 | 1994-10-27 | Sandoz Ltd. | Rapamycin assay |
WO1994025072A1 (en) * | 1993-04-23 | 1994-11-10 | American Home Products Corporation | Rapamycin conjugates and antibodies |
-
1998
- 1998-04-09 EP EP98916710A patent/EP0973805A1/en not_active Withdrawn
- 1998-04-09 WO PCT/CA1998/000361 patent/WO1998045333A1/en not_active Application Discontinuation
- 1998-04-09 AU AU70207/98A patent/AU740304B2/en not_active Ceased
- 1998-04-09 CA CA002286311A patent/CA2286311A1/en not_active Abandoned
- 1998-04-09 JP JP54219898A patent/JP2001518784A/en active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1993025533A1 (en) * | 1992-06-05 | 1993-12-23 | Abbott Laboratories | Methods and reagents for the determination of immunosuppressive agents |
WO1994024304A1 (en) * | 1993-04-08 | 1994-10-27 | Sandoz Ltd. | Rapamycin assay |
WO1994025072A1 (en) * | 1993-04-23 | 1994-11-10 | American Home Products Corporation | Rapamycin conjugates and antibodies |
Cited By (18)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000022000A1 (en) * | 1998-10-09 | 2000-04-20 | Isotechnika, Inc. | Methods for the production of antibodies to specific regions of cyclosporine and cyclosporine metabolites |
US6686454B1 (en) | 1998-10-09 | 2004-02-03 | Isotechnika, Inc. | Antibodies to specific regions of cyclosporine related compounds |
US6713266B1 (en) | 1998-10-09 | 2004-03-30 | Isodiagnostika Inc. | Immunoassay method for measuring a cyclosporine and its metabolites |
US7445916B2 (en) | 2004-04-14 | 2008-11-04 | Wyeth | Process for preparing rapamycin 42-esters and FK-506 32-esters with dicarboxylic acid, precursors for rapamycin conjugates and antibodies |
US7625726B2 (en) | 2004-04-14 | 2009-12-01 | Wyeth | Process for preparing rapamycin 42-esters and FK-506 32-esters with dicarboxylic acid, precursors for rapamycin conjugates and antibodies |
WO2006116727A2 (en) | 2005-04-27 | 2006-11-02 | Dade Rehring Inc. | Compositions and methods for detection of sirolimus |
EP1880215A2 (en) * | 2005-04-27 | 2008-01-23 | Dade Behring Inc. | Compositions and methods for detection of sirolimus |
EP1880215A4 (en) * | 2005-04-27 | 2008-05-14 | Dade Behring Inc | Compositions and methods for detection of sirolimus |
US7883855B2 (en) | 2006-07-21 | 2011-02-08 | Abbott Laboratories | Immunosuppressant drug extraction reagent for immunoassays |
US8541554B2 (en) | 2006-07-21 | 2013-09-24 | Abbott Laboratories | Immunosuppressant drug extraction reagent for immunoassays |
US7914999B2 (en) | 2006-12-29 | 2011-03-29 | Abbott Laboratories | Non-denaturing lysis reagent |
US7993851B2 (en) | 2006-12-29 | 2011-08-09 | Abbott Laboratories | Lysis reagent for use with capture-in-solution immunoassay |
US8129127B2 (en) | 2006-12-29 | 2012-03-06 | Abbott Laboratories | Assay for immunosuppressant drugs |
US8221986B2 (en) | 2006-12-29 | 2012-07-17 | Abbott Laboratories | Diagnostic test for the detection of a molecule or drug in whole blood |
US8329415B2 (en) | 2006-12-29 | 2012-12-11 | Abbott Laboratories | Lysis reagent for use with capture-in-solution immunoassay |
US8404452B2 (en) | 2006-12-29 | 2013-03-26 | Abbott Laboratories | Assay for immunosuppressant drugs |
US8440416B2 (en) | 2006-12-29 | 2013-05-14 | Abbott Laboratories | Diagnostic test for the detection of a molecule or drug in whole blood |
US8697365B2 (en) | 2006-12-29 | 2014-04-15 | Abbott Laboratories | Non-denaturing lysis reagent |
Also Published As
Publication number | Publication date |
---|---|
CA2286311A1 (en) | 1998-10-15 |
EP0973805A1 (en) | 2000-01-26 |
AU740304B2 (en) | 2001-11-01 |
AU7020798A (en) | 1998-10-30 |
JP2001518784A (en) | 2001-10-16 |
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