WO1986001539A1 - Method of producing monoclonal anti-idiotypic antibodies - Google Patents
Method of producing monoclonal anti-idiotypic antibodies Download PDFInfo
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- WO1986001539A1 WO1986001539A1 PCT/US1985/001658 US8501658W WO8601539A1 WO 1986001539 A1 WO1986001539 A1 WO 1986001539A1 US 8501658 W US8501658 W US 8501658W WO 8601539 A1 WO8601539 A1 WO 8601539A1
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- receptor
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- idiotypic
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
- C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
- C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
Definitions
- a receptor is defined herein as a molecular structure that interacts with another structure, referred to as a ligand, as part of a biological process.
- Receptors can include, but are not restricted to, enzymes, immunoglobulins, lymphokines, cell surface molecules, attachment sites on viruses and cells, specific binding proteins such as those which bind nucleic acids, hormone binding molecules and metal-binding molecules such as calmodulin.
- a ligand is similarly defined as a structure that reacts with a receptor as defined above. In addition to binding to a receptor, a ligand as defined herein may act as an agonist or as an antagonist with respect to the receptor.
- Antibodies arise when an animal is immunized with a particular antigen.
- the variable regions of such antibodies contain a set of antigenic determinants known as the idiotype which is usually associated with antigen specificity.
- Anti-idiotypic antibodies may arise when an animal is injected with specific idiotypic antibody molecules which have been previously obtained and purified. In such an experiment the animal immunized with the idiotypic antibody produces antibodies directed against the idiotypic determinants of the injected antibody.
- the idiotypic antibodies may then bind to either the antigen or the anti-idiotypic antibodies so produced. Regardless of functional differences, macromolecules having the same binding specificities can also show homologies at their binding sites.
- immunizing an animal with purified idiotypic antibodies raised against an antigen which is a ligand for a biological receptor may raise anti-idiotypic antibodies which bind to both the idiotypic antibodies and the receptor for the ligand (1).
- Anti-idiotype antibodies thus afford one route to functional anti-receptor antibodies, which have been implicated in several auto-immune diseases. At least some of these diseases (1,2) might originate from an antiidiotypic response to antibodies formed against biologically active ligands normally present in vivo, such as insulin or thyrotropin. The likelihood of an antiidiotypic etiology increases if the patients' antibodies are found to be directed at the combining site of a receptor. Although not always the case (3), this has been found to be true in many patients with myasthenia gravis (4), particularly in those who are severely ill. Moreover, it has been shown (1) that experimental myasthenia gravis can be induced in rabbits via the antiidiotypic route.
- an aberrant anti-idiotypic response could have a role in at least some cases of myasthenia gravis in humans.
- Graves disease the specificity of circulating anti-thyroid receptor antibodies is usually directed at the combining site of the thyrotropin receptor (5). These anti-thyroid receptor antibodies are therefore probably anti-idiotypic, directed at idiotypes of anti-thyrotropin antibody.
- Adenosine receptors are also important for several reasons. They are involved in the regulation of blood flow in arteries and arterioles, in particular in the heart. Thus, they are one of the many factors involved in determining blood circulation in this organ. They are also involved in the modulation of nervous impulses and, generally speaking, tend, when activated, to have a calming effect on animals. There is good evidence that these receptors are the targets for caffeine, which seems to reverse the action of adenosine and produce agitation, rather than tranquility. Finally, there is recent evidence that alcoholism may very well be associated with abnormalities in the adenosine receptor. Previous methods for the production and study of antireceptor antibodies required immunizing animals with purified receptors in order to raise the desired antibodies (6,7 ,8).
- Rabbits were then immunized with a specifically purified preparation of anti-BisQ antibodies to elicit a population of antiidiotypic antibodies specific for the binding sites of anti-BisQ.
- a portion of the anti-idiotypic antibodies (12,13) produced in the second set of rabbits crossreacted with determinants on AChR preparations from Torpedo californica, Electrophorus ejectricus and rat muscle.
- determinants on AChR preparations from Torpedo californica, Electrophorus ejectricus and rat muscle.
- several of the rabbits showed signs of experimental myasthenia gravis, in which circulating AChR antibodies are typically found.
- Anti-idiotypic antibodies against the thyrotropin receptor have also been reported (14).
- TSH thyrotropin
- specific antibodies idiotypic
- rats were injected into rabbits which then produced the anti-idiotypic antibodies against the rat anti-TSH antibodies.
- the anti-idiotypic response plays a role in regulating the immune response (12,13,15).
- injection of an antigen elicits, in addition to antibodies to the antigen, other populations that include anti-idiotypic antibodies directed at the combining sites of the antigen-specific antibodies. If the antigen-specific antibodies recognize a ligand of a receptor, then the antiidiotypic antibodies should bind receptor.
- the present invention surprisingly overcomes the aforementioned limitations by providing a novel method for producing anti-idiotype antibodies which depends on the use of hybridoma technology to immortalize the cells which produce the anti-idiotype antibodies.
- a novel method for producing anti-idiotype antibodies which depends on the use of hybridoma technology to immortalize the cells which produce the anti-idiotype antibodies.
- This invention concerns a method for producing a monoclonal anti-idiotypic antibody, preferably one that binds to a receptor or ligand, and more preferably, one that mimics a receptor or ligand.
- the method is an auto-anti-idiotypic method which involves the following steps:
- lymphoid cells e.g. spleen cells
- Suitable antigens include among others a synthetic or naturally occurring molecule capable of binding to a receptor such as an adenosine receptor; acetylcholine receptor (AChR); steroid receptor, e.g. an estrogen, androgen or glucocorticoid receptor; thyroid stimulating hormone (TSH, thyrotropin) receptor; insulin receptor; ⁇ -adrenergic receptor; dopamine receptor, one of the chemotactic receptors of the neutrophil or other receptors as previously defined.
- a receptor such as an adenosine receptor; acetylcholine receptor (AChR); steroid receptor, e.g. an estrogen, androgen or glucocorticoid receptor; thyroid stimulating hormone (TSH, thyrotropin) receptor; insulin receptor; ⁇ -adrenergic receptor; dopamine receptor, one of the chemotactic receptors of the neutrophil or other receptors as previously defined.
- TSH thyroid stimulating hormone
- the antigen can be conjugated to a protein such as keyhole limpet hemocyanin, thyroglobulin or a serum protein, e.g. bovine or rabbit serum albumin.
- a protein such as keyhole limpet hemocyanin, thyroglobulin or a serum protein, e.g. bovine or rabbit serum albumin.
- the receptor of interest can be an adenosine receptor and the antigen the molecule N 6 -carboxypentamethylene adenosine which is conjugated to a protein such as bovine serum albumin, rabbit serum albumin or keyhole limpet hemocyanin.
- the receptor of interest can also be an acetylcholine receptor, and the antigen the molecule BisQ which is conjugated to a protein such as bovine serum albumin, rabbit serum albumin or keyhole limpet hemocyanin.
- An animal is suitably immunized by a conventional method, e.g. by intraperitoneal, intramuscular, intradermal or other mode of injection with a solution containing the antigen and preferably a suitable adjuvant.
- a suitable adjuvant Preferably the injection is repeated with a suitable booster injection after about two to about four weeks, e.g. after about three weeks.
- a suitable time for collecting lymphoid, e.g. spleen cells from the injected animal is at least about three days after the injection, preferably after a booster injection.
- lymphoid cells may be cultured in vitro in the presence of the antigen to effect antibody production (26). Again, the lymphoid cells may be collected at least about three days after contact and immunization with the antigen.
- Hybridoma cells produced by fusing the lymphoid cells and appropriate myeloma cells are screened by immunoassay, preferably an enzyme immunoassay, of the hybridoma culture medium against an immobilized antibody to the antigen and a labeled antibody capable both of binding to the monoclonal anti-idiotypic antibody under appropriate conditions and of being detected.
- immunoassay preferably an enzyme immunoassay
- the hybridoma cells are screened by immunoassay of the hybridoma culture medium against immobilized receptor and a labeled antibody capable both of binding to the monoclonal anti-idiotypic antibody under appropriate conditions and of being detected.
- the hybridoma cells may be separately cultured by conventional methods in an appropriate medium.
- the appropriate medium may be an in vitro cell culture or a suitable animal host.
- a monoclonal anti-idiotypic antibody and in a preferred embodiment one capable of binding to a receptor under suitable conditions, may be produced without the necessity of immunizing the animal with either a previously identified, recovered and purified idiotypic antibody to the antigen or a previously identified, recovered and purified receptor capable of binding the antigen as a ligand.
- a monoclonal anti-idiotypic antibody may be produced which is capable of binding to a receptor such as an adenosine receptor or an acetylcholine receptor or another receptor as defined above.
- the antibody of this invention acts as an agonist or antagonist of the receptor.
- Fig. 1 Illustrates schematically the synthetic route used to prepare the triamcinolone protein conjugate.
- Fig. 2 Illustrates the binding of Torpedo AChR (a) and rabbit anti-BisQ (b) by F8-D5 monoclonal anti-idiotypic antibody.
- Fig. 3 Illustrates inhibition of binding of F8-D5 monoclonal anti-idiotypic antibody to anti-BisQ by Torpedo AChR.
- Fig. 4 Competitive inhibition of binding of [ 3 H]TA to the rat liver cytosol by increasing levels of unlabeled triamcinolone acetonide (O), steroid derivative 17- [C4-carboxy-1-methylbutylidine) bis (oxy)]-9-fluoro118-21-dihydroxypregna-1,4-diene ( ⁇ ), and by the steroid-BSA conjugate ( ⁇ ).
- O unlabeled triamcinolone acetonide
- ⁇ steroid derivative 17- [C4-carboxy-1-methylbutylidine) bis (oxy)]-9-fluoro118-21-dihydroxypregna-1,4-diene
- ⁇ steroid-BSA conjugate
- Fig. 5 Scatchard plot of the binding of [ 3 H] triamcinolone acetonide to Fab fragments of affinity purified anti-steroid antibodies.
- Fab fragments (0.227 micrograms) were incubated with increasing concentrations of [ 3 H]triamcinolone acetonide (0.4-62.5nM) in the presence of 0.01M phosphate buffered saline pH 7.4 containing 0.1% gelatin in a total volume of 200 microliters.
- Total and non-specific binding 50 micromolar triamcinolone acetonide
- Fig. 6 Competitive inhibition of binding of [ 3 H] triamcinolone to the glucocorticoid receptor and to anti-steroid (unprocessed rabbit serum 540). See legend to Table I for the experimental procedure.
- the inhibitors used were triamcinolone acetonide ( ⁇ ), dexamethason ( ⁇ ), corticosterone ( ⁇ ), hydrocortisone ( ⁇ ), progesterone ( ⁇ ), and dihydrotestosterone ( ⁇ ).
- Fig. 7 Sucrose density gradient analysis. Rat liver cytosol ( ⁇ — ⁇ ), rat liver cytosol and 8G11-C6 ( ⁇ - ⁇ .
- FIG. 8 Binding of 8G11-C6 to anti-steroid Fab fragments. See methods for the experimental procedure. 8G11-C6 was partially purified by precipitating with 50% (NH 4 )SO 4 .
- Fig. 9 & 10 The effect of various steroids, steroid conjugates and RSA on the binding of 8G11- C6 to antisteroid-Fab fragments.
- the method used was similar to that described in the experimental procedure; the only difference being that 155 microliters of inhibitor that was diluted serially 2-fold with PBS was added to each well and pre-incubated at room temperature for 10-20 min. Controls with no inhibitor were included. Add 25 ⁇ of 50% (;NH 4 ) 2 SO 4 precipated 8G11-C6 (1.045 micro grams) mix and incubated for 2 hrs at 37°C. Blanks with only PBS were included as well.
- ⁇ triamcinolone acetonide
- ⁇ triamcinolone-RSA
- ⁇ rabbit serum albumin
- the inhibitors used in Fig. 10 were estradiol 173-RSA ( ⁇ ), testosterone-RSA (o), triamcinolone-RSA ( ⁇ ), deoxycorticosterone-RSA ( ⁇ ) and cortisone-RSA ( ⁇ ).
- Fig. 11 Inhibition of binding of 8G11-C6 to antisteroid Fab fragments with rat liver cytosol ( ⁇ ) or partially purified glucocorticoid receptor ( ⁇ ).
- the method used was similar to that described in the experimental procedure. The only difference being that, to 200 ⁇ of rat liver cytosol or partially purified glucocorticoid receptor preparations, that were diluted 2-fold serially in PBS, 1.045 micrograms of 50% (NH ⁇ J ⁇ SO. precipitated 8G11-C6 was added and pre-incubated at room temperature for 15 mins. 200 ⁇ of the incubation mixture was then added to microtiter wells previously coated with 10 ng anti-steroid Fab fragments and incubated for 2 hrs at 37°C.
- FIG. 12 Binding of 8G11-C6 to parti ally purified glucocorticoid receptor .
- 200 microl iters containing either 1 .08 micrograms ( ⁇ ) or 0 .54 micrograms ( ⁇ ) of a partially purif ied receptor preparation in 0 .1M NaHCO were added to the wells of the polystyrene plastic and incubated overnight at 4°c.
- the wells were washed 2x with PBS-Tween and various amounts of a 2-f old seri ally diluted 50% ( NH 4 ) 2 SO 4 precipitated preparation of 8G11-C6 added. See methods section for the methodology employed .
- Fig. 13 Inhibition of binding of 8G11-C6 to partially purified glucocorticoid receptor .
- 200 microliters containing 0.54 micrograms protein of a partially purified receptor preparation in 0.1 M NaHCO 3 pH 9.3 was added to the wells of polystyrene plates. These were incubated for 2 hrs at 37°C. Washed 2x with PBS containing 0.02% Tween. Add 175 1 of inhibitor serially diluted 2-fold and pre-incubate for 20 min at room temperature before adding 0.836 micrograms of 50%
- this invention provides a method for producing a monoclonal anti-idiotypic antibody which involves:
- Suitable animals for use with this auto-anti-idiotypic method include vertebrates such as mice, rats, rabbits, guinea pigs, pigs, goats and cows. Additionally, human and other vertebrate lymphoid cells may suitably be used in embodiments using in vitro immunization.
- This method provides a more direct route than any previously available for obtaining a monoclonal antibody that binds to, or more preferably mimics the biological activity of a receptor or ligand, e.g. as an agonist or antagonist.
- the antigen comprises a naturally occurring or synthetic molecule capable of binding to a receptor. Suitable receptors include an adenosine receptor; acetylcholine receptor (AChR); steroid receptor, e.g.
- the invention is further applicable to receptors for other pharmacologically active low molecular weight molecules, for regulatory macromolecules such as lymphokines, and for infectious agents such as viruses, and to other receptors as defined above.
- the monoclonal anti-idiotypic antibody so produced is capable of binding to the receptor and may additionally act as an agonist or antagonist with respect to the receptor.
- the antigen may be conjugated to a protein such as a serum albumin, e.g. bovine or rabbit serum albumin, or a protein such as keyhole limpet hemocyanin or thyroglobulin, and is preferably injected into the animal with a suitable adjuvant, e.g. complete Freund's adjuvant, or is used for in vitro immunization of lymphoid cells grown in culture (26).
- a suitable adjuvant e.g. complete Freund's adjuvant
- the animal is injected intraperitoneally with a bovine serum albumin ( BSA) conj ugate of 4- ( 2 ' -succinoyloxyethyloxy) -trans3 ,3 ' -bis ( ⁇ -trimethylammonium) azotoluene ( 1) , depicted bel ow .
- BSA bovine serum albumin
- the synthetic compound containing the antigenic defre rminants of interest is a powerf ul agonist of AChR ( 1) .
- the compound containing the antigenic determinants of interest is the naturally-occurring molecule, adenosine .
- monoclonal antibodies to glucocorticoid receptors were obtained by immunizing an animal with a protein conjugate of an appropriate compound.
- the compound containing the antigenic determinants of interest in this case the glucocorticoid receptor triamcinolone (9-fluoro-11 ⁇ , ⁇ 16 , 17, 21-tetrahydroxypregna-1,4-diene ⁇ 3, 20-dione), is conjugated to a suitable protein with a linker moiety, here ketohexanoic acid.
- a linker moiety here ketohexanoic acid
- An effective antibody-raising amount of antigen or antigen-protein conjugate may be provided by about 0.1 ml of a solution containing about 1 mg/ml of the antigen or conjugate, preferably with an adjuvant such as complete Freund's adjuvant.
- an adjuvant such as complete Freund's adjuvant.
- the animal is injected again with a booster injection of a solution of the antigen.
- the booster injection may be repeated.
- lymphoid cells are collected from the animal, for example by sacrificing the animal and collecting the spleen cells.
- lymphoid cells may be contacted with the antigen more directly by culturing the lymphoid cells in vitro in the presence of the antigen (26).
- the lymphoid cells may be collected, again, at least about three days after the immunizing contact with the antigen.
- a series of hybridoma cells is then prepared essentially by the protocol of Kohler and Milstein (17) as modified by Sharon et al. (18), as indicated above.
- Supernatants from the hybridoma culture medium may then be obtained by a replica transfer technique (19) and screened for activity by immunoassay against the antigen or conjugate, an immobilized antibody to the antigen or conjugate (idiotypic) and in -embodiments where the antigen is a ligand to a receptor, against the appropriate receptor.
- the immunoassay also requires a labeled antibody capable both of binding to the antiidiotypic monoclonal antibody under appropriate conditions and of being detected.
- the immunoassay is enzyme-linked (1) using, e.g., peroxidase labeled goat anti-mouse immunoglobulin as the labeled antibody in embodiments for producing the monoclonal antibodies from mouse spleen cells.
- FIG. 3 shows the results of an experiment in which the binding to anti-BisQ of an (NH 4 ) 2 SO 4 -precipitated supernatant of F8-D5 was inhibited by Torpedo receptor. At the highest concentration of receptor used, 67% inhibition was observed; 50% inhibition occurred with about 8 micrograms of inhibitor in the conditions described in Example 4, below. In experiments in which the amount of antibody was decreased five-fold to 2 micrograms per well, 50% inhibition occurred in the presence of 0.9 micrograms of Torpedo receptor. In a similarly designed series of experiments, 50% inhibition of the binding of F8-D5 monoclonal antibody to Torpedo receptor was accomplished with 3 micrograms of purified rabbit anti-BisQ.
- the TA-BSA conj ugate was found to compete with tritiated triamcinolone [ 3 H ]TA for the glucocorticoid receptor . Its apparent Kd at lOnM [ 3 H]TA for the glucocorticoid receptor was found to be 2.5x10 -6 M. This is 100-fold greater than that of the l igand, triamcinolone acetonide which was found to be 1 .5x10 - 8 M under the same assay conditions (Fig, 4).
- the triamcinolone-BSA conjugate was also found to be immunogenic and to induce polyclonal antibodies specific for triamcinolone. Significant titers could be detected in rabbit serum immediately after the second booster injection. These were detected by preciptin tube assay by incubating rabbit serum with various concentrations ranging from 0 .1-1 .0 mg/ml of either triamcinolone-RSA or RSA. Precipitation was only obtained in the presence of triamcinolone-RSA and not with RSA. When pre- immune serum was used as a control , no precipitation was obtained. Portions of the antiserum were then purif ied by affinity chromatography on an AH-Sepharose 4B-triamcinolone column . 40 ml of serum yielded 15-20 mg anti-steroid, that could also precipitate the triamcinolone-RSA complex by the preciptin tube assay as well as by Ouchterlony.
- glucocorticoid receptor As the steroid binding properties of the glucocorticoid receptor and those of the Fab fragments of the affinity purified anti-steroid antibodies were similar, the latter were used to screen by ELISA for anti-idiotypic antibody production in mice. Isolation of anti-idiotypic antibodies to Fab fragments and the subsets that reacted with the glucocorticoid receptor.
- Antibodies cross reacting with the glucocorticoid receptor were selected from among the anti-idiotypes by their ability to deplete glucocorticoid receptor from rat liver cytosol.
- the anti-idiotypes were first immobilized on CNBr-Sepharose anti-mouse beads and incubated with rat liver cytosol. After 2 hrs, the beads were centrifuged down and the cytosol labeled with [ 3 H]TA. To account for non-specific binding of the glucocorticoid receptor to the beads controls were included. In these either no anti-idiotype or an anti-idiotype namely 5B5 that did not interact with the anti-steroid by ELISA was immobilized on the beads.
- Table II Depletion of glucocorticoid receptor from rat liver cytosol. For the experimental procedure used see methods.
- 8G11 was subcloned and the monoclones 8G11-G5 and 8G11C6 were derived from this line. These were partially purified by precipitating in 50% (NH 4 ) 2 SO 4 . From Table II, it can be seen that CNBr-Sepharose anti-mouse beads that had no antibody attached to them and treated with BM containing 1% FCS depleted 5% of [ 3 H]TA from rat liver cytosol when compared to anti-mouse beads treated with PBS.
- Antiroouse beads with the immobilized control cell line 5B5 or 5B5-B6 also depleted 9% and 4%, respectively, of [ 3 H]TA from rat liver cytosol when compared to the control anti-mouse beads that were treated with PBS.
- the hybridoma cell line secreting 8G11-C6 has been deposited with the A ⁇ erican Type Culture Collection, Rockvill e, Maryland under accession number ATCC HB 8708.
- Table III shows that pr elabeling the receptor by preincubating rat liver cytosol with low levels of [ 3 H] TA ( 2 .5 nM) or high levels [ 3 H ]TA ( 13nM-50nM) for 2 hrs at 4°C and then adding it to CNB r-Sepharose 4B antimouse 8G11-C6 does not inhibit the binding of receptor to the beads .
- the epitope recognized by the anti-idiotypic 8G11-C6 therefore might lie near but not in the ligand binding site of the glucocorticoid receptor .
- the binding of 8G11-C6 to Fab fragments was found to be concentration dependent (Fig. 8). Although it was found that the ligand, triamcinolone acetonide, or rabbit serum albumin on their own did not affect the binding of 8G11-C6 to Fab, the steroid conjugate i.e. triamcinolone-RSA did inhibit the binding of 8G11-C6 to Fab (Fig. 9). In addition, to triamcinolone-RSA, other steroid-RSA conjugates such as deoxycorticosterone-RSA and cortisone-RSA were also found to inhibit the binding of 8G11-C6 to the antisteroid-Fab fragments.
- glucocorticoid receptor was partially purified from rat liver cytosol. As can be seen from Fig. 11, preparations of partially purified glucocorticoid receptor also inhibited the binding of 8G11-C6 to Fab, having enhanced activity at comparable dilutions.
- the 8G11-C6 monoclonal antibody was obtained by an auto-anti-idiotypic route, as were the other embodiments of this invention.
- the strategy employed was to use the hapten, triamcinolone, of the glucocorticoid receptor to raise polyclonal antibodies in rabbits and monoclonal antibodies in mice.
- polyclonal idiotypic anti-steroid antibodies were isolated by affinity chromatography. These were used to screen by ELISA for anti-idiotypic antibody formation in mice using a goat anti-mouse peroxidase label.
- This strategy resembles that used to isolate anti-idiotypic antibodies to the acetylcholine receptor (36) .
- the major difference between the two being that purified acetylcholine receptor was used to screen for anti-idiotypic acetycholine activity in the former study whereas in the present study anti-idiotypic glucocorticoid receptor activity was determined with crude rat liver cytosol preparations by a depletion assay and verified with characterization studies. This demonstrated that purified receptor preparations were not necessary either for immunizing or screening.
- anti-idiotypic antibodies that - are cross reactive with insulin receptor (37), chemotactic receptors of the neutrophil (40), B-ader nergic receptor (38,39), Reovirus receptor (41), dopamine receptor (42), and TSH receptor (43) have also been raised by using ligand and/or idiotypic antibodies as antigens.
- triamcinolone was derivatized to triamcinolone ⁇ -ketohexanoic hydroxysuccinimide ester at the 16 and 17 position of the D ring of the steroid, as this modification would affect its binding activity to the receptor the least.
- the triamcinolone protein conjugates that were synthesized from the steroid ester interacted with the glucocorticoid receptor and elicited both idiotypic and anti-idiotypic antibodies.
- the polyclonal idiotypes raised by the triamcinolone-BSA conjugate in rabbits had high affinities for triamcinolone acetonide and steroid binding specificities that resembled those of the glucocorticoid receptor.
- the binding of 8G11-C6 to the anti-steroid was inhibited by triamcinolone-RSA conjugates and amino acids and peptides containing triamcinolone, indicating that this interaction occurred at the combining site of the idiotype.
- the anti-idiotype 8G11-C6 was initially raised to idiotypic antibodies that were specific for triamcinolone. However, the results show the binding of 8G11-C6 to the antisteroid is inhibited by steroid-RSA conjugates rather than by triamcinolone acetonide. As the ligand is a low M.W., organic molecule it will only occupy a small portion of the total combining surface area of the antisteroid. Hence 8G11-C6 will be able to crossreact with the antisteroid even though triamcinolone is present. On the other hand, in the case of the steroid-RSA conjugate almost the total area will be occupied and the binding of 8G11-C6 to the antisteroid will be inhibited.
- the glucocorticoid receptor can be radiolabeled with [ 3 H]TA and its binding and elution profiles monitored. Furthermore, the sucrose density studies demonstrate that 8G11-C6 has a low affinity for the glucocorticoid receptor, it will enable the receptor to be eluted readily from the affinity column thus increasing the yield of undenatured receptor. In fact, the steroid RSA conjugate could be used to elute the receptor from such a column. 8G11-C6 together with other antiidiotypic antibodies that cross-react with the glucocorticoid receptor may be used as probes instead of radiolabeled ligands to identify and characterize the structure and function of the receptor.
- the strategy described here provides a powerful route to anti-receptor antibodies which are likely to be directed at determinants associated with the combining sites of the receptor.
- the antibodies may be obtained in large quantity by culturing hybridoma cells by conventional in vitro or in vivo methods which are well known in the art.
- puri fied receptor is not required for immunization; in fact, these antidiotypic antibodies, presumably, can be used to isolate receptor.
- this invention is also applicable to receptors for other pharmacologically active, low molecular weight molecules, regulatory macroraolecules such as lymphokines, and infectious agents such as viruses.
- Anti-receptor antibodies prepared with the invention may be useful in prevention and treatment of diseases.
- anti-receptor antibodies may be useful as substitutes for pharmacologically active substances such as interferon, interleukin 2, other lymphokines and in vaccines as substitutes for infectious agents. Immunity to infectious agents such as viruses may be induced with antireceptor antibodies. Anti-receptor antibodies may also be useful as cytotoxic reagents for killing tumor cells, as well as normal cells of the body that may prevent acceptance of transplants. The invention can also be used to determine if a structure is a ligand for an unknown receptor. It is also possible that receptors isolated with antidiotypic antibodies of this invention may themselves be used as therapeutic agents in the future.
- the residual oil was diluted with 4 ml of methanol and applied to four preparative TLC plates of Al 2 O 3 (E. Merck, 1.5 mm, F 254) and developed with ethyl acetate-hexane 2:1.
- the bands with Rf 0.7 were scraped off and eluted with methanol to yield, after distillation in vacuo.
- 0.24 g of an orange viscous oil (III) which was used in the next step, i.e. succinylation.
- IV was linked to bovine serum albumin (and to rabbit serum albumin) by the mixed anhydride technique (24) as follows: 56 rag (0;076 mmole) of IV was dissolved in 3.8 ml of DMF. The solution was cooled in an ice salt bath and 20 microliters (15.6 mg, 0.084 mmole) of trin-butylamine was added, followed by 11 microliters (11.5 mg, 0.084 mmole) of isobutylchlorof ormate. The solution was allowed to stand for 30 min in the ice salt bath and was then added in one portion, with stirring, to a cold solution of 213 mg (3.2 x 10 -3 mmole) of bovine serum albumin in 11.5 ml of water.
- the albumin solution had been adjusted to pH 9 with 0.01 N NaOH.
- the solution was kept in the cold bath for three hours; 0.01 N NaOH had to be added periodically to keep the pH near 9. Then the solution was dialyzed exhaustively in the cold against several changes of distilled water. The product was then lyophilized. Approximately 8 molecules of IV were then linked to the albumin carrier, as determined spectrophotoroetrically by measurement of the absorbance of 324 nm (max, 20,000).
- mice were immunized intraperitoneally (i.p.) with 0.1 ml of a solution of BisQ-BSA conjugate (1 mg/ml) (prepared as in Example 1) in complete Freund's adjuvant. Twenty-three days later, the mice were boosted i.p. with the same preparation. After 5 days, the spleen cells were collected and fused with a nonsecreting myeloma line (P3x63-Ag 8.653) (25) essentially by the protocol of Kohler and Milstein (17) as modified by Sharon et al. (18).
- a nonsecreting myeloma line P3x63-Ag 8.653
- Supernatants from hybridoma clones were obtained by a replica transfer technique (19) and screened for activity against BisQ-RSA, rabbit anti-BisQ and AChR (Torpedo) by an enzyme immunoassay (1), using peroxidase-labeled goat anti-mouse immunoglobulin as the second antibody.
- Anti-BisQ is the specifically purified rabbit antibody used in ref. 1.
- Fig. 2 illustrates, the antibody produced by this clone was able to bind AChR of Torpedo as well as spe cifically purified rabbit anti-BisQ (1). This property was retained by clones obtained after two subsequent subclonings by limiting dilution. No attempt can be made to relate the respective binding affinities because of the characteristics of enzyme immunoassay procedures, in general. For example, there is no way to ensure that the same number of determinant sites of receptor and of anti-BisQ adhere to the plastic wells.
- FIG. 3 illustrates, the binding to anti-BisQ of an (NH 4 ) 2 SO.-preci ⁇ itated supernatant of F8-D5 was inhibited by Torpedo receptor. At the highest concentration of receptor used, 67% inhibition was observed; 50% inhibition occurred with about 8 micrograms of inhibitor in the conditions described. In experiments in which the amount of antibody was decreased fivefold to 2 micrograms per well, 50% inhibition occurred in the presence of 0.9 micrograms of Torpedo receptor. In a similarly designed series of experiments, 50% inhibition of the binding of F8-D5 to Torpedo receptor was accomplished with 3 micrograms of purified rabbit antiBisQ.
- the ether layer was decanted, 15 ml of water was added to the residual oil, and the pH adjusted to 3.0 to yield a clear solution.
- the solution was concentrated to one-half volume in vacuo. Crystals appeared and the solution was allowed to stand for several days at 4°C. The crystals were collected and recrystallized from water to yield 75 mg of CPA. An additional 50 mg was recovered from mother liquors. The compound analyzed correctly for the monohydrate.
- Protocols for immunization and fusion were essentially identical to that described previously for BisQ in Example 2. Immunization was effected with the RSA conjugate, but the BSA conjugate is just as satisfactory. Screening was conducted with the BSA conjugate and with a specifically purified rabbit antibody raised by immunization with the BSA conjugate. Specifically purified antibody was prepared in a manner analogous to that described for anti-BisQ antibody (1) except that the immunoadsorbent was N 6 -(carboxypentaraethylene) adenosine linked to aminohexylsepharose (Pharmacia).
- Triamcinolone (0.8 g) and 0.9 g of ⁇ -ketohexanoic hydroxysuccinimide ester were suspended in 16 ml dioxane with stirring, followed by careful addition of 0.3 ml perchloric acid. Stirring was allowed to continue overnight at room temperature yielding a clear solution. The reaction was terminated by neutralizing with Na 2 CO 3 , and the product extracted with 100 ml methylene chloride. After washing with 200 ml H 2 O, the upper water phase was discarded and the lower methylene chloride phase was dried over solid'MgSO 4 . After filtration and evaporation to dryness, the product was chromatographed by TLC using 10% methanol in chloroform as the developing solvent. The product was detected as an ester as before (27) and distinguished from the starting material by its strong UV activity.
- a chloroform solution of the product was passed through a silica gel 60 column (100 g) that had been equilibrated with chloroform. Development was with 7.5% methanol in chloroform, using a slow flow rate. Ten milliliter fractions were collected and examined for product by TLC chromatography on silica gel 60 using 10% methanol in chloroform as solvent. The fractions containing the triamcinolone ⁇ -ketohexanoic-hydroxysuccinimide ester were combined, concentrated by rotary evaporation and purified further by HPLC, using a ZOBOX silica column of 21.2 mmx25cm dimensions.
- the amount of steroid bound was calculated from the E m ax of the steroid ester, which was previously determined to be 1 .2x10 4 at E ma ⁇ 243 nm .
- the results were 18-23 uni ts steroid/mole RSA, 16 units steroid/mole thyroglobulin.
- New Zealand white rabbits were immunized by multiple intraderraal injections of a total of 1 ml of triamcinolone-BSA (2 mg/ml) emulsified with an equal volume of complete Freunds adjuvant or saline. Booster infections were given three weeks later and then at monthly intervals. Animals were bled bi-weekly from the ear vein and the sera stored at 4°C until required.
- AH-Sepharose 4B (1g) was suspended in 100 ml of 0.5N NaCl. After 15 min, the slurry was filtered on a Buchner funnel and washed with 250 ml of 0.5N NaCl and then with 250 ml water. The gel was transferred to a centrifuge tube and washed five times with 10 ml 0.2M NaHCO 3 pH.8.15. After the last wash, the supernatant was discarded and an equal volume of 0.2M NaHCO 3 pH 8.15, was added followed by 58.6 mg triamcinolone Nhydroxysuccinmide ester in 3-5 ml of tetrahydrof uran.
- mice Two female Balb/c mice were immunized i.p. with 0.1 ml of a 1 mg/ml solution of triamcinolone-thyroglobulin conjugate in complete Freunds adjuvant. Three weeks later the mice were boosted i.p. with the same preparation. After another 4 week interval, the animals were boosted a second time i.p. with the same preparation. Four days after the final immunization, one of the mice was splenectomized. 2x10 spleen cells were fused with 2x10 cells of a non-secreting myeloma line (P3x63-Ag 8.653) (31) according to the procedure of Kohler and Milstein (17) as modified by Sharon et al. (18).
- P3x63-Ag 8.653 non-secreting myeloma line
- Supernatants from the hybridomas were obtained by a replica transfer technique (32) and screened for antiidiotypic activity by ELISA.
- Monoclonal antibody-producing hybridomas were obtained by cloning the cells of the cultures of interest on soft agar or by using a raicroraanipulation technique (33).
- the class and subclass of the heavy chain of the monoclonal antibodies were determined by Ouchterlony and by ELISA using antimouse Ig class and subclass antisera as typing serum or to coat microtitre plates.
- the clones of interest were expanded by growing them in 75 cm 2 T-flasks.
- the antibodies in the culture medium were purified by precipitation in 50% saturated (NH 4 ) 2 SO 4 .
- the presence of anti-idiotypic antibodies in the hybridoma culture medium were assayed by a double antibody sandwich ELISA.
- Polystyrene microplates (Corning 25855) were coated by adding 200 microliters of 50 ng/ml affinity purified rabbit anti-steroid FAB fragments in 0.1M NaHCO 3 , pH 9.3, to the wells and incubating overnight at 4°C. After washing twice with 0.01M phosphate buffer - 0.14M NaCl, pH 7.4, containing 0.05% Tween (PBS-tween), the culture medium from the hybridomas was added and incubated at 37°C for 2 hrs.
- PBS-tween 0.01M phosphate buffer - 0.14M NaCl, pH 7.4, containing 0.05% Tween
- the wells were washed three tiroes with PBS-tween and 200 microliters of 1:3,000 dilution in PBS-tween of goat anti-mouse IgM-IgG horseradish peroxidase (Tago) was added. After incubating 1 hr at 37°C, the wells were washed three times with PBS-tween and 200 microliters substrate (7 mg o-phenylenediamine dichloride in 10 ml 0.1M citrate-phosphate buffer, pH 4.8 containing 5 microliters of 30% H 2 O 2 ) was added. Depending on the intensity of the color, the reaction was stopped after 5-10. min by the addition of 50 microliters 8N H 2 SO 4 and the OD determined (Titretek) at 492 nm.
- livers were then removed, homogenized with a Teflon-glass homogenizer in 1.1 volume of 10 mM HEPES, containing 50 mM NaCl, 1 mM Na 2 EDTA, 1 mM dithiothreitol (DTT) and 10% glycerol, pH 7.6, and centrifuged for 1 hr at 250,000 g at 4°C. The upper fatty layer was discarded.
- the supernatant was either frozen immediately in liquidnitrogen and used for assays or labeled by incubating with 75 nM [ 3 H] triamcinolone acetonide for 2 hrs at 4°C. unbound steroid was removed with 5% dextran-coated charcoal.
- the [ 3 H]TA glucocorticoid receptor was partially purified by the method described by Gametchu & Harrison (9), the only modification being that the phosphocellulose was omitted from the first purification step.
- Affinity chromatography-purif ied rabbit or goat antimouse 1gM was coupled to cyanogen bromide activated Sepharose-4B as described by Westphal et al. (35) and in the Affinity Chromatography Handbook of Pharmacia.
- Cyanogen bromide (CNBr)-activated Sepharose 500 mg was swollen in ImM HCl and washed on a sintered funnel with 1 mM HC1 (200 ml). The gel was washed twice with coupling buffer (0.25 M NaHCO 3 containing 0.5M NaCl pH 8.5) and suspended immediately in a (4 mg/3 ml) affinity purified anti-mouse IgM solution.
- Antibody containing culture medium (1-3.5 ml) was mixed with 125 microliters of anti-mouse-Sepharose-4B beads overnight at 4°C.
- the AM-Sepharose-4B beads were centrifuged down and washed 3 times with 2 ml PBS.
- Rat liver supernatant (300 microliters) containing 0.090.18 praoles of receptor was added and mixed for 2 hrs at 4°C.
- the gel was centrifuged down and 200 microliters of the supernatant was assayed for glucocorticoid receptor after adding 25 microliters of 25nM
- [ 3 H]TA was removed by adding 100 micrograms of 5% charcoal/dextran and allowing it to stand at 4°C for 5 mins. Cold PBS (1 ml) was added and after 5 mins the charcoal/dextran was centrifuged down. The supernatant was asssayed in a scintillation counter.
- the glucocorticoid receptor was radiolabeled by incubating 0.5 ml rat liver cytosol with 10nM [ 3 H]TA for 2 hrs at 4°C. Five percent charcoal/dextran (0.25 ml) was added and the suspension allowed to stand for 10 min at 4°C. After centrifugation, 4°C, either 50 microliters of [ 3 H]TA radiolabeled cytosol (0.16 p moles) was incubated together with 190 microliters of PBS at 4°C, or 50 microliters of [ 3 H]TA radiolabeled cytosol together with 190 microliters of 50% (NH 4 ) 2 SO4 precipitated 8G11 C6 (9.4 mg/ml).
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Abstract
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US64455084A | 1984-08-27 | 1984-08-27 | |
US644,550 | 1984-08-27 |
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WO1986001539A1 true WO1986001539A1 (en) | 1986-03-13 |
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PCT/US1985/001658 WO1986001539A1 (en) | 1984-08-27 | 1985-08-27 | Method of producing monoclonal anti-idiotypic antibodies |
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EP (1) | EP0191846A4 (en) |
JP (1) | JPS62500421A (en) |
AU (1) | AU4777385A (en) |
WO (1) | WO1986001539A1 (en) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1987005703A1 (en) * | 1986-03-19 | 1987-09-24 | Boehringer Mannheim Gmbh | Antiparatypical antibodies, their use for detecting and treating auto-immune diseases |
EP0250119A2 (en) * | 1986-06-20 | 1987-12-23 | Vasocor | Atherosclerotic anti-idiotype antibody immunoassay and reagents |
EP0334687A1 (en) * | 1988-01-26 | 1989-09-27 | Yeda Research And Development Company Limited | Immunoassays for incipient diabetes |
EP0344211A1 (en) * | 1987-02-20 | 1989-12-06 | Imclone Systems, Inc. | Monoclonal antibodies in vaccine formulations |
US4925787A (en) * | 1987-09-08 | 1990-05-15 | Agency Of Industrial Science And Technology | Monoclonal anti-idiotypic antibody, method for production thereof, and hybridoma producing said antibody |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CA1341277C (en) * | 1987-03-16 | 2001-07-31 | Roland Keith Mcgready | Anti-paratopic antibody and a method of its manufacture |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4513088A (en) * | 1983-03-30 | 1985-04-23 | The Board Of Trustees Of The Leland Stanford Junior University | Assay for monoclonal antibody against surface Ig of a human B cell tumor |
-
1985
- 1985-08-27 EP EP19850904390 patent/EP0191846A4/en not_active Ceased
- 1985-08-27 WO PCT/US1985/001658 patent/WO1986001539A1/en not_active Application Discontinuation
- 1985-08-27 JP JP50391785A patent/JPS62500421A/en active Pending
- 1985-08-27 AU AU47773/85A patent/AU4777385A/en not_active Abandoned
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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US4513088A (en) * | 1983-03-30 | 1985-04-23 | The Board Of Trustees Of The Leland Stanford Junior University | Assay for monoclonal antibody against surface Ig of a human B cell tumor |
Non-Patent Citations (10)
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1987005703A1 (en) * | 1986-03-19 | 1987-09-24 | Boehringer Mannheim Gmbh | Antiparatypical antibodies, their use for detecting and treating auto-immune diseases |
EP0250119A2 (en) * | 1986-06-20 | 1987-12-23 | Vasocor | Atherosclerotic anti-idiotype antibody immunoassay and reagents |
EP0250119A3 (en) * | 1986-06-20 | 1990-04-18 | Vasocor | Atherosclerotic anti-idiotype antibody immunoassay and reagents |
EP0344211A1 (en) * | 1987-02-20 | 1989-12-06 | Imclone Systems, Inc. | Monoclonal antibodies in vaccine formulations |
EP0344211A4 (en) * | 1987-02-20 | 1990-03-12 | Imclone Systems Inc | Monoclonal antibodies in vaccine formulations. |
US4925787A (en) * | 1987-09-08 | 1990-05-15 | Agency Of Industrial Science And Technology | Monoclonal anti-idiotypic antibody, method for production thereof, and hybridoma producing said antibody |
EP0334687A1 (en) * | 1988-01-26 | 1989-09-27 | Yeda Research And Development Company Limited | Immunoassays for incipient diabetes |
Also Published As
Publication number | Publication date |
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JPS62500421A (en) | 1987-02-26 |
EP0191846A1 (en) | 1986-08-27 |
AU4777385A (en) | 1986-03-24 |
EP0191846A4 (en) | 1988-04-27 |
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