IE911172A1 - MONOCLONAL ANTI-IgM ANTIBODIES, THEIR PRODUCTION AND USE,¹AND HYBRIDOMAS FOR PRODUCING THE SAME - Google Patents

Abstract

A principal aspect of the invention concerns rat monoclonal antibodies that: (a) bind selectively to IgM sub-type antibodies; (b) are IgGs; (c) do not bind to IgG1 or IgG2 sub-type. The preferred embodiment of these antibodies is one designated 2G10, and functional equivalents thereof.

Description

This invention is in the fields of immunology and monoclonal antibody production. More particularly it concerns monoclonal anti-IgM antibodies, hybridomas that produce these antibodies, immunochemicals made from those antibodies, and the use of those immunochemicals.

BACKGROUND OF THE INVENTION Since the report of Kohler and Milstein describing the production of monoclonal antibodies, the development of technology to produce immortalized lymphocytes capable of producing antibodies of predetermined specificity has had a major impact on both clinical and basic scientific research as well as the therapeutic modalities available for the diagnosis and treatment of a wide variety of pathological conditions.

Antibodies are endogenous proteins produced by the immune system in response to antigenic stimuli. These proteins specifically bind to antigen molecules at defined sites (epitopes). Polyclonal antibodies are derived from immunization of animals with antigens and they bind to these antigens at multiple sites (epitopes). Monoclonal antibodies, on the other hand, are a specific, defined set of antibodies which are derived from a single clone (monoclone) of cells producing a specific antibody. In contrast to polyclonal antibodies monoclonal antibodies bind to only one specific epitope on the antigen molecule. -2Although the technology for the generation of monoclonal antibodies has existed for some time, the current methodology is time-consuming, laborious and often results in the production of antibodies which although specific for the target antigen, are of relatively low affinity for the antigen, and thus are of limited usefulness in a wide variety of applications.

Among the difficulties encountered in the production of useful and clinically relevant monoclonal antibodies is the abundance of antibodies of the IgM sub-type obtained from hybridomas produced by standard in vivo or in vitro immunization procedures. The IgM sub-type antibodies are generally of low affinity, are difficult to purify and often comprise the bulk of antibodies produced by hybridomas. In addition, in mixed cultures of IgM and IgG secreting hybridoma cells, IgM secreting cells often overgrow the IgG secreting hybrid cells.

Part of the laborious procedure for the production of hybridomas is the elimination of the IgM producing hybridoma cells produced after a cell fusion. This is generally done by cloning the cells by limiting dilution, growing up the individual cells into colonies, and testing each colony individually to determine which colonies produce IgG sub-type antibodies. Generally, the IgG producing hybridoma cells are then further analyzed to determine the antigen specificity of the antibodies produced.

Although antibodies have been reported which are directed against epitopes on the IgM antibody, all tested to date were also reactive with IgG sub-type antibodies.

Linking cytotoxic agents to antibodies to make immunotoxins has been disclosed by the applicants and others. Recent interest has centered on immunotoxins of monoclonal antibodies conjugated to the enzymatically active portions (A chains) of toxins of bacterial or plant origin via hetero-bifunctional agents. Nevelle, D.M. and Youle, R. J., Immunol Rev (1982) £2.: 75-91; Ross, W.C.J., et al., -3European J Biochem (1980) 104; Vitteta, E.S., et al., Immunol Rev (1982) £>: 158-183; Raso, V., et al., Cancer Reg (1982) 42: 457-464; Trowbridge, I.W. and Domingo, D.L., Haturs,ί-Cgnd) (i98i) iw 171-173.

SUMMARY QF THIj-lMVENUON A principal aspect of the invention concerns rat monoclonal antibodies that: (a) bind selectively to IgM sub-type antibodies; (b) are IgGs; (c) do not bind to IgGx or IgG2 sub-type.

The preferred embodiment of these antibodies is one designated 2G10, and functional equivalents thereof.

The rat x rat hybridomas that produce the above described antibodies and progeny of those hybridomas are other aspects of the invention.

The invention also includes a method of preparing a hybridoma as defined above comprising fusing rat tumor cells with rat splenocytes obtained from a rat immunized with murine IgM sub-type immunogen and selecting for hybridomas producing antibody as defined above.

A further aspect of the invention is a method of producing antibody as defined above comprising culturing a hybridoma having the ability to produce such antibody, or optionally a hybridoma which has been prepared by effecting a method as claimed above.

Another aspect of the invention relates to immunotoxins and their preparation by conjugating (a) the above described monoclonal antibodies, and (b) a cytotoxic moiety or magnetic beads.

Another aspect of the invention concerns labeled derivatives of the above described monoclonal antibodies that are labeled with a detectable label that permits the derivatives to be used in targeting, specific selection or sorting.

Another aspect of the invention concerns a method of killing IgM producing hybridoma or B cells by contacting the ^911172 -4cells with a cytocidally effective amount of one or more of the above described immunotoxins.

Other aspects of the invention are direct and indirect immunoassays for determining whether a cell is producing IgM antibodies or to determine whether an antibody is of the IgM isotype. These assays involve incubating the cells with the monoclonal antibodies or labeled derivatives thereof. When the labeled derivatives are used, the presence of labeled binary immune complexes on the cells as read directly. When unlabeled antibody is used the cells are further incubated with a labeled antibody against the monoclonal antibody and the presence of labeled ternary immune complexes on the cells is read.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 demonstrates screening of hybridoma supernatants for IgM-specific antibody.

Figure 2 demonstrates the dose-dependent specific binding of antibody 2G10.

Figure 3 demonstrates the effect of increasing absorbed murine immunoglobulin content on 2G10 binding.

Figure 4 shows selective recognition of murine IgM by 2G10 in an indirect ELISA.

Figure 5 illustrates the purity of 2G10 antibody by SDS-PAGE..

Figure 6 characterizes the subclass of rat 2G10 antibody by Ouchterlony immunodiffusion.

Figure 7 demonstrates utilization of 2G10 for binding to cells expressing IgM subclass antibodies.

Figure 8 illustrates the elution profile of immunotoxin (composed of 2G10 coupled to gelonin) on a gel filtration matrice.

Figure 9 demonstrates the purity of 2G10-gelonin immunotoxin by SDS-PAGE.

Figure 10 shows the specific binding of immunotoxin(2G10-gelonin) to murine IgM. -5DETAILED DESCRIPTION OF THE INVENTION In order that the invention herein described may be more fully understood, the following detailed description is set forth.

As used herein the term monoclonal antibody means an antibody composition having a homogeneous antibody population. It is not intended to be limited as regards the source of the antibody or the manner in which it is made.

As used herein with respect to the exemplified rat 10 monoclonal anti-murine IgM antibodies, the term functional equivalent means a monoclonal antibody that: (a) crossblocks an exemplified monoclonal antibody; (b) binds selectively to murine IgM antibody; (c) has a G isotype; and (d) does not bind to IgGx or IgG2 isotype.

As used herein with regard to the monoclonal antibody-producing hybridomas of the invention the term progeny is intended to include all derivatives, issue, and offspring of the parent hybridoma that produce the monoclonal anti-murine IgM antibody produced by the parent, regardless of generation or karyotypic identity.

The present invention may be utilized to produce antibodies that will bind to IgM antibodies of any species. It is only necessary to utilize the teaching of the present invention to obtain a hybridoma cell line which is stable and continues to produce the anti-IgM antibody directed to the immunizing specie. Preferably the anti-IgM monoclonal antibody of the present invention is directed to a murine or human IgM.

Monoclonal Antibody Production The antibody-producing fusion partners that are used to make the hybridomas of this invention are generated by immunizing rats with murine IgM antibody. The rats are inoculated subcutaneously and intraperitoneally with an immunogenic amount of the murine IgM antibody in Feund's adjuvant and then boosted with similar amounts of the immunogen in adjuvant. Spleens are collected from the •Ε 911172 -6immunized rats a few days after the final boost and a cell suspension is prepared therefrom for use in the fusion.

Hybridomas are prepared from the splenocytes and a rat tumor partner using the general somatic cell hybridization technique of Kohler, B. and Milstein, C., Nature (1975) 256: 495-497 [as modified by Buck, D. W., et al, In Vitro (1982) W 377-381]. Available rat myeloma lines, such as YB2/0 and Y3-Ag 1.2.3, may be used in the hybridization.

Basically, the technique involves fusing the tumor cells and splenocytes using a fusogen such as polyethylene glycol.

After the fusion the cells are separated from the fusion medium and grown in a selective growth medium, such as HAT medium, to eliminate unhybridized parent cells. The hybridomas are expanded, if desired, and supernatants are assayed for anti-murine IgM activity by conventional immunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay) using the immunizing agent IgGj, IgG2 and IgM (murine IgM antibody) as antigen. Positive clones are characterized further to determine whether they meet the criteria of the invention antibodies.

Hybridomas that produce such antibodies may be grown in vitro or in vivo using known procedures. The monoclonal antibodies may be isolated from the culture media or body 25 fluids, as the case may be, the conventional immunoglobulin purification procedures such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration, if desired.

Monoclonal Antibody Selection/Characterization 30 The important characteristics of the monoclonal antibodies are (1) their immunoglobulin class, (2) their selectivity for murine IgM antibody, and (3) their usefulness in identifying and binding to murine IgM producing hybridoma cells.

The selectivity and range of a given antibody is determined by testing it against panels of (1) IgGlf IgG2 -7and IgM producing hybridoma cells and (2) IgGx, IgG2 and IgM antibodies. In selecting the claimed antibodies approximately 162 growing hybridoma cultures were initially screened. Nine clones reacted with the murine IgM antibody but not IgG. One of these clones was chosen for further characterization.

Antibodies exhibiting acceptable selectivity and range were conjugated to gelonin using N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) or iminothiolane (IT) as a coupling agent. The conjugates were tested against IgM and IgG coated plates (Figure 11) to determine if specificity of the antibody is preserved after chemical coupling to the toxin.

Further details of the characterization of this antibody are provided in the examples below.

The immunochemical derivatives of the monoclonal antibodies of this invention that are of prime importance are immunotoxins (conjugates of the antibody and a cytotoxic moiety and labeled (e.g., radiolabeled, enzyme-labeled, magnetic-labeled or fluorochrome-labeled) derivatives in which the label provides a means for identifying and/or sorting immune complexes that include the labeled antibody.

The cytotoxic moiety of the immunotoxin may be a cytotoxic drug or an enzymatically active toxin of bacterial or plant origin, or an enzymatically active fragment (A chain) of such a toxin. Enzymatically active toxins and fragments thereof are preferred and are exemplified by gelonin, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phvtoiacca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, mitogellin, restrictocin, phenomycin, and enomycin. Gelonin is most preferred. Conjugates of the -8monoclonal antibody and such cytotoxic moieties may be made using a variety of bifunctional protein coupling agents. Examples of such reagents are SPDP, IT, bifunctional derivatives of imidoesters such as dimethyl adipimidate 5 HCl, active esters such as disuccinimidyl suberate, aldehydes such as glutaraldehyde, bis-azido compounds such as bis(p-azidopenzoyl) hexanediamine, bis-diazonium derivatives such as bis-(p-diamoniumbenzoyl)ethylenediamine, diisocyanates such as tolylene 10 2,6-diisocyanate, and bis-active fluorine compounds such a 1,5-difluoro-2,4-dinitrobenzene.

When used to kill murine IgM antibody producing hybridomas in vitro, the conjugates will typically be added to the cell culture medium at a concentration of at least about 10 nM. The formulation and mode of administration for in vitro use are not critical. Agueous formulations that are compatible with the culture or perfusion medium will normally be used. Cytotoxicity may be read by conventional techniques to determine the presence or degree of IgM producing hybridoma cells.

When used in vivo for suppression of IgM producing cells, the immunotoxins are administered to the immunized animals in therapeutically effective amounts (i.e., amounts that eliminate or reduce the IgM producing splenocytes).

They will normally be administered parenterally, preferably intravenously. The dose and dosage regimen will depend upon the nature of the IgM producing cell to be suppressed, the characteristics of the particular immunotoxin, e.g., its therapeutic index, and onset of action. The amount of immunotoxin administered will typically be in the range of about 0.1 to about 10 mg/kg of body weight.

For parenteral administration, the immunotoxins will be formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle. Such vehicles are inherently nontoxic and nontherapeutic. Examples of such vehicles are -9water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils and ethyl oleate may also be used. Liposomes may be used as carriers. The vehicle may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives. The immunotoxin will typically be formulated in such vehicles at concentrations of about 1 mg/ml to 10 mg/ml.

Cytotoxic radiopharmaceuticals for eliminating IgM 10 producing hybridoma cells may be made by conjugating high linear energy transfer (LET) emitting isotopes (e.g., Y, Pt) to the antibodies. The term cytotoxic moiety as used herein is intended to include such isotopes.

The labels that are used in making labeled versions of 15 the antibodies include moieties that may be detected directly, such as fluorochromes and radiolabels, as well as moieties, such as enzymes, that must be reacted or derivatized to be detected. Examples of such labels are 32p, 123j, 3H, i4C, fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferia, 2,3-dihydrophthalazinediones, horseradish peroxidase, alkaline phosphatase, lysozyme, and glucose-6-phosphate dehydrogenase. The antibodies may be tagged with such labels by known methods. For instance, coupling agents such as aldehydes, carbodiimides, dimaleimide, imidates, succinimides, bis-diazotized benzadine and the like may be used to tag the antibodies with the above-described fluorescent, chemiluminescent, and enzyme labels. The antibodies may also be labeled with magnetic beads for use in magnetic sorting regimens.

The antibodies and labeled antibodies may be used in a variety of cell sorting procedures to separate the IgM producing hybridoma cells from IgG producing hybridoma cells or to eliminate the IgM producing hybridoma cells from cultures containing such cells. -10Common assay techniques that may be used include direct and indirect assays. Direct assays involve incubating a hybridoma or antibody of unknown isotype with a labeled antibody of the present invention. If the sample includes IgM producing cells, the labeled antibody will bind to those cells. After washing the cells to remove unbound labeled antibody, the sample is read for presence of labeled immune complexes. In indirect assays the cell sample is incubated with unlabeled monoclonal antibody. The sample is then treated with a labeled antibody against the monoclonal antibody (e.g., a labeled anti-rat antibody), washed, and read for the presence of labeled ternary complexes.

For diagnostic use or assays to determine the presence of IgM isotype the antibodies will typically be distributed in kit form. These kits will typically comprise: the antibody in labeled or unlabeled form in suitable containers, reagents for the incubations and washings, labeled anti-rat antibody if the kit is for an indirect assay, and substrates or derivatizing agents depending on the nature of the label. IgM antigen controls and instructions may also be included.

The following examples provide a detailed description of the preparation, characterization, and use of a representative monoclonal antibody of this invention. These examples are not intended to limit the invention in any manner.

EXAMPLE 1 Source and Characterization of Rat anti-Mouse IgM Monoclonal Antibodies A rat hybridoma designated 58.6 was obtained from Dr.

Joanne Trial, Department of Immunology, M.D. Anderson Cancer Center. Originally, the 58.6 cell line secreted a rat antibody. The cell line was not stable and after about four subcultures, the 58.6 cells ceased producing any antibody.

An original stock of cells was then cloned by limiting 11dilutions to obtain a cell line that would be stable and continue producing anti-IgM antibodies.

A) Cloning by limiting dilution 58.6 cells were cultured in Iscove's medium for 3 days 5 at 37°C in a humidified atmosphere of 5 CO2 in air. When the expanded cell culture had reached 50% confluency, cells were harvested by centrifugation and counted using a hemacytometer. Cells were diluted in 50% fresh medium and 50% conditioned medium (medium in which 58.6 cells had been grown for 7 days) and plated at approximately one cell per well into 96 well plates. When wells containing single cells had grown to small colonies (approximately 12 days), the medium was removed and assayed for anti-IgM antibody as described in Example 2. Positive cells were expanded for large scale production of antibody, freezing of cell stocks, and further characterization of the antibody. When cell lines were fully characterized for the type and specificity of antibody produced, appropriate cell lines were recloned and expanded for freezing of cells stocks and injections into pristane-treated nude mice for production of ascites fluid.

B) Freezing of hybrid cells When hybrid cells plated into T75 flasks had reached 70% confluency, cells were collected by centrifugation and the pellet was resuspended in 0.9 ml of fetal bovine serum. Immediately before freezing, the cells were transferred to freezing vials and 0.1 ml of dimethyl sulfoxide were added to each vial. The vials were stored in liquid nitrogen.

C) Ascites fluid production Approximately 107 hybridoma cells were washed in serum-free media and injected intraperitoneally into nude mice which had received an intraperitonea1 injection of 0.5 ml of pristane 7 to 14 days earlier. Ascites fluid usually formed within 1-3 weeks and was collected from the peritoneal cavity using a large gauge needle. Fluid was collected into tubes containing 5 ml of PBS with 20 mM EDTA. -12After centrifugation at 2000 x g for 10 min, the supernatant was saved, made 0.1% in sodium azide, and stored at 4°C or frozen at -20°C in small aliguots. This fluid provided a rich source of monoclonal antibody (approximately 5-10 mg/ml).

Example 2 Selection and Assay of Hybridoma Cells Producing Rat Anti-mouse IoM Antibodies Hybridoma colonies which grew to a density of approximately 500-1000 cells within 2 weeks were chosen for further analysis. In order to determine which of the hybridoma cells produced antibodys which bound murine IgM antibody. Hybridoma culture medium from these colonies was assayed for the presence of rat anti-mouse IgM by the Enzyme-linked immunosorbent assay (ELISA) performed according to the procedure of Voller et al.(ref). 100 mg of mg/ml of purified mouse IgM or IgG protein (Sigma Chemical Company, St. Louis, Mo.) was diluted into coating buffer (50 mM NaHCO3, pH 9.8) and absorbed overnight onto 96-well microtifer plates by incubation at 4°C in a humidified chamber. The wells were then washed three times with phosphate-buffered saline containing 0.2% Tween-20 (PBS-Tween). After washing and removal of all traces of liquid in the wells by tapping lightly onto paper towels, 100 ug of hybridoma supernatant was added to the IgM-coated wells and incubated at room temperature for 2 hours. Plates were again washed with PBS-Tween, and incubated for one hour at room temperature with 100 ul of a 1:1000 dilution of peroxidase conjugated goat anti-rat IgG in PBS-Tween. After the wells were washed again as described above, they were reacted with 100 ul of 1 mM ABTS (2,2-azino-di(3-ethylbenzthiazoline sulphonic acid) in 0.1 M sodium citrate buffer pH 4.2, containing 0.03% hydrogen peroxide) for 20-60 min at 37°C. Optical density was measured at 405 nm on a MicroElisa Reader. -13In order to assess whether the epitope recognized by the anti-IgM antibodies were on the heavy chain or light chain of the antibody, the binding of 9 hybridoma supernatants to IgM lambda and IgM-kappa protein coated plates was tested. Hybridomas IC2 and 2G10 both bound equally well to IgM-kappa and IgM-lambda coated wells, indicating that the binding specificity of the anti-IgM antibodies was on the heavy (mu) chain of the IgM antibody. That the other 7 antibodies tested also bound to murine IgM is indicated by the results shown on Figure 1. As can be seen in Figure IA and IB, nine different hybridomas were tested against either IgM-kappa or IgM-lambda coated plates. A standard ELISA assay was performed to measure rat immunization bound to each plate. All antibodies were found to bind to both IgM-k and IgM-1 coated wells. Among the highest binding antibodies, 1C2 and 2G10 were chosen for further study. Because of its growth and antibody production characteristics, antibody 2G10 was finally selected. This gave an optical density of 0.3 ± 0.03 (standard deviation). The background was 0.06 optical density units (O.D.) using media without rat monoclonal antibody. Wells that gave a reaction on the anti-murine IgM antibody of greater than or equal to 0.3 O.D. were saved.

The specificity of the rat monoclonal 2G10 antibody was tested by ELISA under a variety of conditions. To determine the selective recognition of IgM vs. IgG by 2G10 lOOng of mouse IgM-lambda protein or IgG3-lambda protein was absorbed onto microtiter plates (100 ul) and incubated with increasing concentrations of 2G10 antibody. As shown in Figure 2, in the antibody dose range of 2-1000 ng, the 2G10 bound to IgM-coated microtiter plates to a greater extent than that achieved in IgG-coated plates. At 2G10 antibody doses of 50 ng and below, no binding was measureable on IgG-coated plates whereas IgM-coated wells were recognized by 2G10. -14The effect of increasing immunoglobulin coating concentration on microtiter plates and recognition by antibody 2G10 was also examined. As shown in Figure 3, 2G10 antibody (added at a dose of 100 ng in 100 ul) incubated in IgG3 and IgM-coated wells shows a selective binding to IgM-coated plates at all coating concentrations. Ten-fold greater reactivity was measureable by ELISA on IgM-versus IgG-coated plates at a coating dose of 10000 ng of IgM or IgG (compare IgG3-kappa to IgM-kappa). The 2G10 antibody again demonstrates selective IgM binding.

Antibody 2G10 was also tested for its ability to selectively recognize IgM in an indirect ELISA assay. Antigens were coated to microtiter plates and were incubated with hybridoma culture media which contained antibody which recognizes these antigens. Antibodies of the IgM and IgGl subclass were used in this assay. After incubation of antigen-coated plates with their interacting antibody, increasing concentrations of rat 2G10 antibody were added to the wells and the binding of 2G10 was evaluted by ELISA. As shown in Figure 4, 2G10 sensitively and selectively recognized IgM bound to its respective antigen (closed circles), but was unable to detect IgG (open circles) under the same conditions, although the presence of IgGl could be easily detected with antibodies reactive with all mouse immunoglobulin subtypes. These results demonstrate the selective recognition of mouse IgM subclass immunoglobulins by 2G10 rat monoclonal antibody. The rat monoclonal antibody was also able to recognize IgM antibody at picogram doses, demonstating its high affinity of murine IgM antibody.

Example 3 Characterization of Rat Anti-Mouse IgM Antibodies Mouse immunoglobulin of various subtypes (IgM-kappa and lambda, IgGx, IgG2, and IgG3) were coated on microtiter plates as in Example 2 and the binding of the rat anti-mouse IgM antibodies produced by the hybridoma cells which were -15positive in Example 2 were further characterized. All plates were read in a Bio-Tek ELISA plate reader at a wavelength of 405 nm. Absorbance (compared to controls) was used an an indication of the presence of antibody against mouse IgM.

Example 4 Purification and characterization of ?giq rat mcngcicnfli aatibgdy A representative rat anti-mouse IgM monoclonal antibody, designated 2G10, was chosen for further characterization. As was shown in Example 3, the 2G10 antibody was positive for IgM kappa and lambda. The antibody was purified by centrifugation and ammonium sulfate fractionation.

The representative hybridoma cell line designated 2G10 was deposited with the American Type Culture Collection (ATCC), Rockville, Md., U.S.A., on April 23, 1990 and assigned Deposit Accession No. HB 10436. The deposits are available pursuant to the patent laws and regulations of the United States and of those countries foreign to the United States in which counterparts of this application are filed. The availability of the deposit does not constitute a license to practice the invention of this application in derogation of any patent issued thereon or on any division or continuation of this application. 2G10 culture supernatant (or ascites fluid from nude mice) was made 45% saturated in ammonium sulfate content (salting out) by the slow addition of an equal volume of 90% saturated ammonium sulfate solution. The sample was stirred for 30 minutes at 4°C and then centrifuged at 20,000 x g for minutes. The pellet was resuspended in a 40% saturated ammonium sulfate solution, stirred 30 minutes and repelleted by centrifugation as described above. The pellet was resuspended in water and dialyzed against 100 volumes of PBS. Aliquots of the solution was used for determination of protein content (by optical density at 280nm), purity (by -16SDS-PAGE) and binding specificity (by ELISA). The remaining antibody solution was frozen at -20°C until needed.

IgM antibodies were purified by ammonium sulfate precipitation and gel filtration on a 2.6 x 40 cm column of chromatographic resin containing agarose, dextran and/or acrylamide eluting with PBS/0.01% sodium azide at room temperature at a flow rate of 1 ml/min.

The subclass of 2G10 rat monoclonal antibody was determined by the method of Ouchterlony (Ouchterlony and Nilsson (1958) in Handbook of Exp. Immun. Weir, ed., Blackwell Scientific, London, ppl9.1-19.44) using an immunodiffusion kit commercially available through ICN Immunobiologicals. (Lisle, IL.).

The subclass of antibody 2G10 is important in evaluating how to purify this molecule. To perform subclass analysis, an Ouchterlony immunodiffusion technique kit was employed. Briefly, antisera against various rat immunoglobin sub-types was added to each of the satellite wells. In the center well, a known standard or unknown sample was added and allowed to diffuse into the semisolid media. A precipitation band at the site of the specific antisera indicates the subtype. As shown in Figure 6, the positive control samples containing all rat sub-type antibodies shows reaction lines at all of the sub-type wells. On the other hand, 2G10 antibody reacted only with the IgG2a sub-type antisera which designates that rat antibody 2G10 is an IgG2, antibody.

In order to evaluate the binding of a commercially-available rat antibody to mouse IgM, the ELISA reactivity of rat monoclonal antibody LO-MM-9 (from Serotec, cat#MCA 199) against murine IgM was evaluated. One hundred nanograms of mu-k, gamma-k, or gamma-1 was added to each well of a 96 well plate. Various ammounts of LO-MM-9 rat antibody were then added and an ELISA assay for rat antibody was performed as described previously. As shown in Table 1, -17there was no binding of this rat antibody to murine IgM coating the wells.

Table 1 Evaluation of commercially available rat antibodies against murine IgM ELISA.-Esastivity against . umr-ine lg Ppngantra-tion Mu-k gamaa-H gamma-1 looo ng 0.058 0.008 0.037 500 ng 0.034 0.004 0.031 100 ng 0.032 0.001 0.021 50 ng 0.030 0.001 0.020 10 ng 0.021 0.001 0.002 5 ng 0.018 0.001 0.024 1 ng 0.009 0.001 0.020 0.1 ng 0.020 0.001 0.023 Thus, this antibody was not deemed useful for further study. In addition, as shown in Figure 6, lane 3, this antibody preparation contained at least three major protein bands and at least five minor protein bands as assessed by SDS PAGE.

Example 5 Binding of Rat Anti-Mouse IgM to IgM Producing gslis In order to demonstrate that the 2G10 rat anti-mouse IgM antibody bound not only to purified IgM antibody coating a 96 well plate but also to cells producing an IgM antibody, FACS analysis was performed on 10C1 cells and murine 238-57 ADR hybridoma cells which secrete IgG and IgM respectively. Briefly, 1x10® cells were centrifuged at 500 x g for 3 min., washed three times with PBS and resuspended in 3 ml of PBS.

Fluorescein conjugated affinity purified F(ab)2 fragment goat anti-mouse immunoglobin IgM (Cappel) was diluted 1:100 in PBS (lx) and 20-40 ul was added to a 20 ul cell suspension. After incubation for 15-20 minutes in the -18dark at room temperature, the cells were washed twice with PBS centrifuging at 500 rpm for 3 minutes.

An aliquot of 300 ul of paraformaldehyde (1% in PBS) is added to fix the cells. The cells were incubated at 4°C until sorted by flow cytometry.

For indirect staining, hybridoma cells are first incubated with rat anti-mouse IgM antibody 2G10, washed, then stained with the fluorescein F(ab)2 fragment goat anti-mouse immunoglobulin IgM and sorted by flow cytometry.

As shown in Table 2 and Figure 7, antibody 2G10 bound specifically to IgM present on the surface of IgM secreting murine hybridoma cells and not to IgG secreting cells.

Table 2 FACS Analysis of IgM and IgG Expressing Murine Hvbridoma Cells % of Fluorescein-labeled cells IgG IgM TREATMENT_HYBRIDS_HYBRIDS 0 0.87 0.37 Irrelevant Rat IgG + FITC Goat Anti-Rat 0.23 0.96 2G10 Rat Anti-Mouse IgM AbY + FITC Goat Anti-Rat 1.58 89.99 As Table 2 shows, there was no background fluorescein of untreated cells. Irrelevant rat IgG also did not bind to either IgG hybrids or IgM hybrids. There was no binding of the 2G10 antibody to IgG producing hybridoma cells (1.58% of cells positive. Figure 7A and Table 2). However, as shown in Figure 7B and Table 2, 90% of cells producing murine IgM antibody were shown to bind strongly to the 2G10 antibody. Therefore, the binding of antibody 2G10 to cells occurs due to the recognition of the murine IgM on the cell surface.

,E 911172 -19It will be appreciated that the properties of the antibodies examined are effectively the only relevant characteristics of the corresponding hybridomas in that, for the purposes of the present invention, the hybridomas are characterized by their ability to produce particular antibodies having said properties.

Cvtotoxicitv Evaluation The claimed antibodies were conjugated to ricin toxin A chain (RTA) treated with SPDP as described by Carlsson, J., et al, Biochem J (1978) 173; 723-737 or with iminothiolane (IT).

Example 6 coupling of 2G10 to Gelonin A stock solution of SPDP reagent (N-Succininidyl 3-(2-pyridylditho) proprionate) (6mg/ml) in dry DMF was prepared. To 1 ml of a PBS solution containing 1 mg of 2G10 antibody in a 12 x 75 mm glass test tube, SPDP was slowly added to a 5-fold molar excess (approx. 10 ul of stock solution). The mixture was vortexed every 5 minutes for 30 minutes at room temperature.

Excess unreacted SPDP was removed from the sample by gel filtration chromatography on a Sephadex G-25 column (lx 24 cm) pre-equilibrated in 100 mM sodium phosphate buffer pH 7.0 containing 0.5 mM EDTA (Buffer A). Fractions (0.5 ml) were collected and analyzed for protein content using the Bradford dye binding assay (Bradford, (1976) Anal. Biochem. 72; 248-254). Absorbance (600 nm) was monitored in a 96-well plate using a Bio-TEK Microplate autoreader.

Antibody eluted at the void volume (fractions 14-20) and these fractions were pooled and kept at 4°C.

Gelonin toxin was extracted from the seeds of gelonium multiflorum and purified to homogeneity utilizing the method of Stirpe, et al (Stirpe et al., J, Biol. Chem, 255: 6947-6953 (1980)). One milligram of purified gelonin (2 mg/ml in PBS) was added to triethanolamine hydrochloride (TEA/HCl) buffer to a final concentration of 60 mM TEA/HCl ¢911172 -20and adjusted to pH 8.0. The solution was made 1 mM EDTA. 2-iminothiolane stock solution (0.5M in 0.5M TEA/HCl pH 8.0) was added to a final concentration of 1 mM and the sample was incubated for 90 minutes at 4°C under nitrogen gas.

Excess 2-iminothiolane reagent was removed by gel filtration on a column of Sephadex G-25 (1 x 24 cm) pre-equilibrated with 5 mM bis-tris acetate buffer pH 5.8 containing 50 mM NaCl and 1 mM EDTA. Fractions (0.5 ml) were collected and analyzed for protein content in 96 well microtiter plates using the Bradford dye binding assay.

Gelonin eluted in fractions 14-20 and these fractions were pooled and stored at 4°C. SPDP-modified antibody 2G10 was mixed with a 5-fold molar excess of 2-iminothiolane modified gelonin. The pH of the mixture was adjusted to 7.0 by the addition of 0.05 M TEA/HCl buffer (pH 8.0) and the mixture was incubated for 20 hrs at 4°C under nitrogen.

Iodoacetamide (0.1M in PBS) was added to a final concentration of 2 mM to block any remaining free sulfhydryl groups and incubation was continued for an additional hour at 25°C.

Purification of 2G10 Gelonin Complexes To remove low molecular weight products and non-conjugated gelonin, the reaction mixture was applied to a Sephadex S-300 column (1.6 x 31 cm) previously equilibrated with PBS. Fractions (1.0 ml) were collected and 50 ul aliquots were analyzed for protein content using the Bio-Rad dye binding assay. To remove unconjugated 2G10, the high molecular peak (fraction 17-23) from the S-300 column was applied to an affinity chromatography column of Blue Sepharose CL-6B (l x 24 cm) pre-equilibrated with 10 mM phosphate buffer (pH 7.2) containing 0.1 M NaCl. After sample loading, the column was washed with 30 ml of buffer to completely elute non-conjugated antibody. The column was eluted with a linear salt gradient of 0.1 to 2 M NaCl in 10 mM phosphate buffer pH 7.2. Protein content of the eluted -21fractions was determined by the dye-binding assay described previously.

The coupling mixture containing free 2G10 antibody, 2G10 gelonin and free gelonin was first purified by gel filtration on an S-300 column. As shown in Figure 8, a high molecular weight peak was detected (fractions 25-42 )as well as a lower molecular weight peak (fractions 55-67).

Fractions 26-42 were pooled for analysis of conjugate purity and reactivity.

PAGE analysis of the purified 2G10 gelonin conjugate was performed. As can be seen in Figure 9, the 2G10 gelonin coupling mixture (lane 3) contained free 2G10, free gelonin (arrows) as well as 2G10 coupled to 1 gelonin molecule (monomer arrow) and 2G10 coupled to 2 gelonin molecules (dimer, arrow ). As seen in Lane 1, the final purified 2G10 gelonin conjugate contains mostly 2G10 coupled to 1 gelonin molecule and lesser amounts of 2G10 coupled to 2 gelonin molecules. The preparation was not contaminated by free gelonin or free antibody.

To determine whether the chemical reaction and coupling of this 2G10 antibody to gelonin modified the recognition of the 2G10 antibody for murine IgM, 96 well plates were coated with either murine IgG or IgM antibody as in Figure 2. Instead of 2G10 antibody, the 2G10 gelonin conjugate was added to wells at various concentrations. A standard ELISA assay was then performed to detect rat antibody. As shown in Figure 10, the 2G10 gelonin conjugate bound readily to IgM and only to a small extent to IgG coated plates. Only at the highest concentration tested (1000 ng/well) did the 2G10 gelonin conjugate bind significantly to the IgG-coated wells. In contrast, at a similar concentration the 2G10 gelonin conjugate bound extensively to the IgM coated wells. Thus, the immunoreactivity of the 2G10 gelonin conjugate towards IgM was preserved. In addition, the 2G10 gelonin conjugate failed to cross-react appreciably with murine IgG and therefore the selectivity of the 2G10 gelonin conjugate -22was also unaltered. This data suggests that the 2G10 gelonin conjugate should bind to IgM coating murine cells in the same manner as that of antibody 2G10 itself.

Example 7 Production of ODC Monoclonal Antibodies A. In Vitro Immunization and Monoclonal Antibody Production Protocols employed currently for the production of murine monoclonal antibodies result in the generation of hybridomas which secrete antibodies of the IgM subclass. In order to demonstrate the typical result of murine monoclonal antibody production protocols, two techniques were employed for generation of monoclonal antibodies against a rat protein ornithine decarboxylase (ODC). The first technique involves immunization of murine spleen cells in vitro, i.e., in culture dishes, with ODC protein.

This technique was performed by the method of Luben (Luben and Mohler, (1980) Molec, Immunol. 12:635-639) using 25 ug of purified rat liver ODC.

Briefly, ODC protein was incubated with mouse cells for hours at 37°C in the presence of thymocyte conditioned medium (a source of immunoglobulin secreting cell type specific growth factors). The spleen cells were then fused with MPC myeloma cells using polyethylene glycol as a fusogen. The resultant hybrids cells were tested for the secretion of antibody reactive with ODC protein. The results are summarized on Table 3. «=9^72 -23Table 3 IN VITRO IMMUNIZATION AND HYBRIDOMA PRODUCTION SUMMARY Prvqwrtlgn Summary Remarks Total wells plated Total wells with viable hybrids/Total wells plated Total wells positive by first ELISA Wells positive for ODC activity precipitation/ wells tested Total wells expanded for cloning Total individual ELISA positive monoclones recovered from 12 original polyclonal wells Total monoclonals positive for ODC activity precipitation/ Total assayed Total monoclones secreting IgM/ total monoclones Total monoclones secreting IgG/total monoclones Two 24-well plates. x 106 cells/well 48/48 Represents a minimum fusion frequencey of It 10® 48/48 14 of these were greater than 4-fold above control media in ELISA 12/14 Only the 14 most positive in ELISA were tested 12/48 Limited to the 12 most promising clones As few as one recovered from polyclonal well and as many as 18 S0/70 As much as 48% of the added ODC activity precipitated and as little as 4% 70/70 0/70 ---------24After initial screening with ODC protein, the hybridomas were recloned and retested. Seventy hybridomas were obtained which reacted with the ODC protein by several criteria. However, as shown on Table 3, all of the clones were secreting IgM isotype antibodies, none were secreting IgG antibodies. These antibodies were found to be of limited use in a variety of applications for monoclonal antibodies.

B. In Vivo immunization and Monoclonal Antibody 10 Production.

A second technique employed for the production of monoclonal antibodies was the immunization of a mouse by injection with purified ODC protein. Mouse spleen cells were isolated after immunization, fused with P3 X 63 Ag 8.653 myeloma cells (utilizing PEG as described above).

Clones were isolated, tested for reactivity with ODC protein and further characterized for utilization as a specific ODC recognizing reagent. The results of these tests are shown in Table 4. -25Table 4 IN VIVO IMMUNIZATION AND INTRASPECIES HYBRIDOMA PR9PUCTIQH SUMMARY Production Summary Remarks Total wella plated Total wella with viable wella hybrida Total wella poaitive eignifiby firat ELISA 15 control Wella cloned by limiting dilution Total individual ELISA positive monoclones recovered from 30 original polyclonal wells Total monoclonea secreting IgM number tested Total monoclones secreting IgG number tested Total monoclonea precipitating 30 OOC activity/number tested 1200 Numerous wella of 96-well plates plated with 2.5 x 10s cella/well 148 Approximately 12% of plated (about It 107 fusion frequency). /148 Six of these were cantly higher than in binding to ODC in the ELISA All poaitive polyclonea expanded and cloned Three polyclonal wella yielded no poaitive monoclonea 27/27 No comment 0/27 1/5 Only one precipitated a significant amount of ODC activity over control media •Ε 911172 -26Twenty-seven monoclonal cell lines were developed, 100% of which secreted antibody of the IgM subclass. Again these antibodies were later found to be inadequate for utilization as ODC-specific reagents.

Example 8 FACS (Flow Activated Cell Sorting) Procedure IgM secreting hybridoma cells 238-57 ADR were centrifuged at 500 x g for 3 min., washed three times with PBS and resuspended in 3ml of PBS.

Fluorescein conjugated affinity purified F(ab)2 fragment goat anti-mouse immunoglobulin IgM (Cappel) is diluted 1:100 in PBS (lx) and 20-40 ul is added to 20 ul cell suspension.

After incubation for 15-20 minutes in the dark at room temperature, the cells are washed twice with PBS centrifuging at 500 rpm for 3 minutes.

An aliquot of 300 ul of paraformaldehyde (1% in PBS) is added to fix the cells. The cells were incubated at 40°C until sorted by flow cytometry.

For indirect staining hybridoma cells are first incubated with rat anti-mouse IgM antibody 2G10, washed, then stained with the fluorescein F(ab)2 fragment goat anti-mouse immunoglobulin IgM and sorted by flow cytometry.

Example 9 Magnetic Cell Separation Mouse hybridoma cells 238-57 ADR are washed three times in Iscove's medium containing 1% fetal bovine serum and 0.1% gentamicin. The cells are centrifuged at 500 x g for three minutes at room temperature and then counted. The pellet is resuspended in 0.5 ml medium and incubated for 60 minutes on ice with purified rat anti-mouse IgM antibody (2G10) at a concentration of approximately 0.425 mg/ml (about 0.1 ml is used per 106 cells/ml). After washing twice in cold Iscove's medium, the cells are resuspended in 0.2 ml medium and counted.

Magnetic beads are washed 3 times in serum-free medium using a magnetic board. The cell pellet is mixed with the bead pellet at a ratio of 20 beads/cell. The total volume should not exceed -270.4 ml. The cell/bead mixture is incubated for 1/2 hour on ice, agitating every 10 minutes.

The cell/bead mixture is resuspended in at least 2 ml of medium and separate magnetically perpendicular to gravity. Once separation is complete, the supernatant is removed without disturbing the magnetic pellet. The beads are resuspended in 1-2 ml medium and observed miscroscopically.

The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the invention as set forth below.

Claims (19)

1. A monoclonal antibody which: (a) binds selectively to IgM antibody; (b) does not bind to IgG x or IgG 2 antibody; and 5 (c) has a G isotype.

2. A rat monoclonal antibody which: (a) binds selectively to murine IgM antibody; (b) does not bind to IgG x or IgG 2 antibody; and (c) has a G isotype. 10

3. A monoclonal antibody according to either of claims 1 or 2 which binds to hybridoma cells producing anti-IgM antibody.

4. A monoclonal antibody according to either of claims 1 or 2 which binds to hybridoma cells producing anti-mouse IgM antibody.

5. A monoclonal antibody according to any of claims 1-3, inclusive, which is produced by one of the following hybridomas: (a) 2G10; (b) 1C2; or a monoclonal antibody which is functionally equivalent to any one of the aforesaid antibodies.

6. A rat X rat hybridoma which produces a monoclonal antibody according to any of claims 1-5, inclusive, and progeny of said hybridoma.

7. A hybridoma which is: (a) HB 10436; or progeny thereof. IE 9111 72 -298. An immunotoxin which is a conjugate of a monoclonal antibody according to any one of claims 1 to 3 and a cytotoxic moiety.

8. 9. A method of killing murine IgM antibody producing 5 cells comprising contacting said cells with a cytocidally effective amount of an immunotoxin as defined in claim 8.

9. 10. A monoclonal antibody according to any one of claims 1 to 3 and labeled with a detectable label.

10. 11. A method for collecting hybridoma producing IgG 10 isotype monoclonal antibodies comprising treating a hybrid cell population with any one of the antibodies of claims 1-3, inclusive, subjecting said resulting immuncomplexed cells to sorting in a cell sorter, and collecting the cells which have not complexed with said antibodies. 15

11. 12. A method of making an immunotoxin comprising conjugating antibody as defined in claim 1 with a cytotoxic moiety.

12. 13. A monoclonal antibody according to claim 1, substantially as hereinbefore described and exemplified.

13. 14. A rat X rat hybridoma accordint to claim 6, substantially as hereinbefore described and exemplified.

14. 15. An immunotoxin according to claim 8, substantially as hereinbefore described and exemplified.

15. 16. A method according to claim 11 for collecting a hybridoma producing IgG isotype monoclonal antibodies, substantially as hereinbefore described and exemplified.

16. 17. A hybridoma producing IgG isotype monoclonal antibodies, whenever collected by a method claimed in claim 11 or 16 . - 30

17. 18. A method according to claim 12 of making an immunotoxin, substantially as hereinbefore described and exemplified .

18.

19. An immunotoxin whenever made by a method claimed in claim 12 or 18.