CA2079901A1 - Monoclonal anti-igm antibodies, their production and use, and hybridomas for producing the same - Google Patents
Monoclonal anti-igm antibodies, their production and use, and hybridomas for producing the sameInfo
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- CA2079901A1 CA2079901A1 CA002079901A CA2079901A CA2079901A1 CA 2079901 A1 CA2079901 A1 CA 2079901A1 CA 002079901 A CA002079901 A CA 002079901A CA 2079901 A CA2079901 A CA 2079901A CA 2079901 A1 CA2079901 A1 CA 2079901A1
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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
- C07K16/4283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6873—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting an immunoglobulin; the antibody being an anti-idiotypic antibody
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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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
WO~1~17257 PCTJUS91/02822 MONOCLONAL ANTI-I~M ANTIBODIES, ;~
THEIR PRODUCTION AND US~,_AND HYBRIDOMAS FOR
PRODUCING THE SAME ~
FIELD OF_THE INVENTION ~-This invention is in the fields of immunology and monoclonal an~ibody production. More particularl~ it concerns monoclonal anti-IgM antibodies, hybridomas that produce these antibodies, immunochemicals made from those 10 antibodies, and the use of those im~unochemicals.
~ACKGROUND OF THE INVEN~IO~ -Since the report of Kohler and Milstein describing the production of monoclonal antibodies, the development of technology to produce immortalized lymphocytes capable o~
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 condi'cions.
Antibodies are endogenous proteins produced by the immune system in response to antigenic stimuli. These proteins specifically bind to antigen molecules at de~ined sites ~epitopes). Polyclonal antibodies are derived from immunization of animal~ 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) o~ cells producing a specific antibody. In contrast to polyclonal antibodies monoclonal antibodies bind to only one specific epitope on the antigen molecule. ;~
WO91~17257 PCT/US91/02~22 3 ~, l Although the technology for the generation of monoclonal antibodies has existed for some time, the current methodology is time-con~iuming, 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 hy 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 than 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 I'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 ~ia hetero-bifunctional agents. Nevelle, D.M. and Youle, R.
J., Immunol Rev (1982) 62: 75-9l; Ross, W.C.J., et al., W09lt17257 PCT/US9l/02822 -European J Biochem (1980) 104; Vitteta, E.S., et al., Immunol Rev (1982) 62: 158-183; Raso, v., et al., cancer Res (1982) 42: 457-464; Trowbridge, I.W. and Domingo, D.L., Nature tCond) (1981) 294: 171-173.
SUMMARY OF THE INVENTION
A principal aspect o~ the invention concerns rat i, monoclonal antibodies that:
(a) bind selectively to IgM sub-type antibodies;
(b) are IgGs;
(c) do not bind to IgGl or IgG2 sub-type.
The preferred embodiment of these antibodies is on~
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 khe 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 muxine 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 immunoti~xins 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 '~, .
' ~
WO91/17257 P~TIVS91/02822 cells with a cytocidally e~fective amount of one or more of the above described immunotoxins.
Other aspects of the invention are direct and indirect ~-immunoassayis 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 o~ 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 l demonstrates screening of hybridoma i~
supernatants for IgM-specific antibody.
Figure 2 demonstrates the dose-dependent specific binding of antibody 2~10.
Figure 3 demonstrates the ef~iect of increasing absorbed murine immunoglobulin content on 2Gl0 binding.
Figure 4 shows selective recognition of murine IgM by 2Gl0 in an indirect ELISA.
Figure 5 illustrates the purity of 2Gl0 antibody by SDS-PAGE.
Figure 6 characterizes the subclass of rat 2Glb antibody by Ouchterlony immunodiffusion.
Figure ~ demonstrates utilization of 2Gl0 for binding to cells expressing IgM subclass antibodies.
Figure 8 illustrates the elution profile of immunotoxin (composed of 2Gl0 coupled to gelonin) on a gel filtration matrice. . `
Figuxe 9 demonstrates the purity of 2Gl0-gelonin immunotoxin by SDS-PAGE.
Figure l0 shows the specific binding of ~r~unotoxin(2G10-ge1On1n) to rurine IgM.
' .. .
~.
wos1~172s7 ~ r~,.,3 ~ P~r/USg1/0~822 DETAILED DESCRIPTION OF THE INVENTION
In order that the invenkion herein described may be more fully understood, the following deta~lqd 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 i~ made.
As used herein with respect to the exemplified rat monoclonal anti-murine IgM antibodies, the term "~unctional 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 IgG1 or IgG2 isotype. ~;
As used herein with regard to khe 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. Pre~erably 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 IgN antibody. The rats are inoculated subcutaneously and intraperitoneally with an immunogenic amount o~ the murine IgM antibody in ~eund's adjuvant and then boosted with similar amounts of the ;~
immunogen in adjuvant. Spleens are collected from the ", ' .: ,, ... .. , ... ,. . : , . - ~ : . , . . , : . : :: . . .
W~9~/17257 PCT/US91tO2822 O' ,,., ,", ; ..., ., ~., --6-- ;
immunized 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) 18: 377-3al]. 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 IgGl, IgG2 and IgM (murine IgM antibody) as antigen. Positive clones are characterized further to ~O determine whether they meet the criteria of the invention antibodies.
Hybridomas that produce such antibodies may be grown n Yitro or in v vo using known procedures. The monoclonal antibodies may be isolated from the culture media or body 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 SeIection/Characterization The important characteristics of the monoclonal antibodies are (1) their immunoglobulln class, (2) their sel~ctivity for murine Ig~ antibody, and ~3) their usefulness in identifying and binding to murine IgM 7 producing hybridoma cells.
The selectivity and range of a given antibody is detexmined by testing it against panels of (1) IgGl, IgG2 , ,;
: `:
, 2, ~ J 1.
and IgM producing hybridoma cells and (2) IgGl, IgG2 and IgM .,.
antibodies. In selecting the claimed antibodies approximately 162 growing hybridoma cultures were initially screaned. 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 1~-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) or iminothiolane (IT) as a coupling agent. The conjugates were tested against IgM and IgG coated plates (Figure ll) to determine if specificity of the antibody is preserved after chemical coupling to the toxin.
Further details of the characterization o~ this antibody are provided in the examples below.
Immunochemicals 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 ~ragments thereof are preferred and are exemplified by gelonin, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas ~ ), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuri~es ~ordii 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 ' .~;i.. . . . , -. ... ".. . .. . . , , , ~, .. .. . ; . . . .
WO91/17257 ~.~ pCT/US91/02822 monoclonal 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 o~ imidoesters such as dimethyl adipimidate -HCl, active esters such as disuccinimidyl suberate,aldehydes such as glutaraldehyde, bis-a2ido compounds such as bis(p-azidopenzoyl) hexanediamine, bis-diazonium derivatives such as bis-(p-diamoniumbenzoyl)-ethylenediamine, diisocyanates such as tolylene 2,6-diisocyanate, and bis-active fluorine compounds such a l,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 lO nM. The formulation and mode of administration for ln vitro use are not critical. Aqueous ~ormulations 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 n 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 ~he range o~
about O.l to about lO 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 .: ';
,'. :~
`!' ' . ' ' ~ ; . , ; :
Wo91/1~257 PCT/US9~/028~2 2~ '~'~''3~
_g water, saline, Ringer's solution, dextrose solution, and 5%
human serum albumin. Nonaqueous vehicles such as fix~d 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 l mgtml to lO mg/ml.
Cytotoxic radiopharmaceuticals for eliminating IgM
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 the antibodies include moieties that may be detected directly, such as fluorochromes and radiolabels, ~s well as moieties, such as enzymes, that must be reacted or derivatized to be detected. Examples of such labels are 32p, ~25I, 3H, l4C, fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luci~eria, 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 antibodie~ may also be labeled with magnetic beads for use in magnetic sorting regimens.
The antibodies and labeled antibodies may be u~ed in a variety of cell sorting procedures to separate the IgN
producing hybridoma cells from IgG producing hybridoma cells or to eliminate the IgM producing hybridoma cells from cultures containing such cells.
~ , .
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wO91/l7257 PCT/US9l/02822 Common 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 ;~
-~ith 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 lab~led or unlabeled form in suitable containers, reag~nts for tbe 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.
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 `-~:
' `, .' ,:'' ~
WO91/172~7 2~ PCT/US91/02822 dilutions to obtain a cell line that would be stable and continue producing anti-IgM antibodies.
A) Cloning by limiting dllution 58.6 cells were cultured in Iscovels medium for 3 days at 37C 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 h~macytometer. 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, a~d 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 ~or freezing of cells stocks and injections into pristane-treated nude mice for production of ascites fluid.
B) Freezing of hybrid cells When hy~rid cells plated into T75 flasks had reached 70% confluency, cells were collected by centrifugation and the pellet was rèsuspended in 0.9 ml o~ fetal bovine serum.
Immediately before freezing, the cells were transferred to `
freezing vials and O.l ml of dimethyl sulfoxide were added to each vial. The vials were stored in liquid nitrogen.
C) Ascites ~luid production Approximately 107 hybridoma cells were washed in serum-free media and injected intraperitoneally into nude mice which had received an intraperitoneal injection of 0.5 ml of pristane 7 to 14 days earlier. Ascites fluid usually formed within 1-3 weeks and waæ collected from the peritoneal cavity using a large gauge needle. Fluid was collected into tubes containing 5 ml of PBS with 20 mM EDTA. ~;
:
:
WO91/17257 PCT/U~1/02822 ~ t; . . ~ - 12-A~ter centrifugation at 2000 x g for l0 min, the supernatant was saved, made 0.1% in sodium azide, and stored at 4C or frozen at -20C in small ali~uots. This fluid provided a rich source of monoclonal antibody (approximately 5-l0 mg/ml).
Example 2 Selection and Assav of Hvbridoma Cells Producin~ Rat Anti-mouse IqM Antibodies ~ybridoma colonies which grew to a density of approximately 500-lOO0 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 ~rom these colonies was as~ayed 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). l00 mg of l 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 4C in a humidified chamber. The wells were then washed three times with phosphate-buffered saline containing 0.2% Tween-20 ;
(PBS-~ween). After washing and removal of all traces of liquid in the wells by tapping lightly onto paper towels, l00 ug o~ hybridoma supernatant was added to the IgM-coated wells ~nd incubated a~ room temperature for 2 hours. Plates were again washed with PBS-Tween, and incubated for one hour at room temperature with l00 ul of a l:l000 dilution of peroxidase conjugated goat anti-rat IgG in PBS-Tween. After the wells were washed again as described above, they were reacted with l00 ul o~ 1 mM ABTS ~ ~`
(2,2-azino-di(3-ethylbenzthiazoline sulphonic acid) in 0.l M
sodium citrate buffer pH 4.2, containing 0.03% hydrogen ~;
peroxide) for 20-60 min at 37C. Optical density was measured at 405 nm on a NicroElisa Reader.
.. : ~ . .. :; .. : . . , . , , , ,,. .. , : ~ . . .. .
WO91/17257 ~ PCT/US91/02822 In 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-~appa protein coated plates was tested. Hybridomas IC2 and 2GlO 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.
~hat the other 7 antibodies tested also bound to murine IgM
is indicated by the results shown on Figure l. As can be seen in Figure lA and lB, nine different hybridomas were tested against either IgM-kappa or IgM-lambda coated plates.
A sta~dard ELISA assay was performed to measure rat immunization bound to each plate. All antibodies were found to bind to both IgM-k and IgM-l coated wells. Among the highest binding antibodies, lC2 and 2GlO were chosen for further study. Because of its growth and antibody production characteristics, antibody 2GlO 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 TgM
antibody of greater than or equal to 0.3 O.D. were saved. ~ ~`
The specificity of the rat monoclonal 2GlO antibody was tested by ELISA under a variety of conditions. To determine the selective recognition of IgM vs. IgG by 2GlO lOOng of mouse IgM-lambda protein or IgG3-lambda protein was absorbed onto microtiter plates (lO0 ul) and incubated with increasing concentrations of 2GlO antibody. As shown in Figure 2, in the antibody dose range of 2-lO00 ng, the 2GlO
hound to IgM-coated microtiter plates to a greater extent than that achieved in IgG-coated plates. At 2GlO antibody doses of 50 ng and below, no binding was measureable on IgG-coated plates whereas IgM-coated wells were recognized by 2GlO.
WO 91/17257 PcI`/US91/o2822 J`'J ~' -14-The effect of increasing immunoglobulin coating concentration on microtiter plates and recognition by antibody 2G10 was also examin~d. 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 IqM-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 indixect 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, ;~, incxeasing concentrations of ra~ 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 ko its respective antigen (closed circles), but was unable to detect IgG (open circles) under the same conditions, although the presence of IgG1 could be easily detected with antibodies reactive with all mouse immunoglobulin subtypes. These results demonstrate the selective recognition of mouse IgM subclass immunoglobulins by ~G10 rat monoclonal antibody. The rat monoclonal ;~
antibody was also able to recognize IgM antibody at picogram doses, demonstating its high a~finity of murine IgM
antibody.
Example 3 Characterization of Rat Anti-Mouse IgM Antibodies Mouse immunoglobulin of various subtypes (IgM-kappa and lambda, IgG1, 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 .
WO91/17257 ~ PCT~US9l/02822 positi~e 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 2GlO rat monoclonal antibody A representative rat anti-mouse IgM monoclonal antibody, designated 2GlO, was chosen for further characterization. As was shown in Example 3, the 2GlO
antibody was positive for IgM kappa and lambda. The antibody was purified by centrifugation and ammonium sulfate fractionation.
~he representative hybridoma cell line designated 2GlO
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. ~he deposits are available pursuant to the patent laws and regulations of the United States and of those countries foreign to the United S~ates 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.
2GlO 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 sul~ate solution. The sample was stirxed for 30 minutes at 4C and then centrifuged at 20,000 x g for 30 minutes~ The pellet was rssuspended 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 lO0 volumes of PBS. Aliquots of the solution was used for determination of protéin content (by optica1 density at 280nm), purity (by .
W~91~17257 PCT/US91/02822 ~ 16-SDS-PAGE) and binding speci~icity (by ELISA). ~he remaining antibody solution was frozen at -20C until needed.
IgM antibodies were purified by ammonium sulfate precipitation and gel ~iltration on a 2.6 x 40 cm column o~
chromatographic resin containing agarose, dextran andtor acrylamide eluting with PBS/0.01~ sodium azide at room temperature at a flow rate o~ 1 ml/min.
The subclass of 2G10 rat monoclonal antibody was determined by the method of Ouchkerlony (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.).
~he subclass of antibody ~G10 is important in evaluating how to purify this molecule. To perform subclass analysis, an Ouchterlony immunodiffuæion 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-l 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, '~
' : -~:
' .. :......... .. . .
W09tJ1725~ PCT/US91tO2822 ~r~' there was no binding of this rat antibody to murine IgM
coating the wells.
Table 1 Evaluation of commercially available rat antibodies 5against murine IgM
ELISA reactivity aaainst murine_Ia oncentration ~ gamma-k qamma-l lOOO ng 0.058 00008 0.037 500 ng 0.034 0.004 0.031 :~ -~
10 100 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 15 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 20 SDS PAGE.
Example 5 Bindina of Rat Anti-Mouse IqM to IaM Producinq Cells In order to demonstrate that the 2G10 rat anti-mouse :
IgM antibody bound not only to purified IgM antibody coating 25 a 96 well plate but also to cells producing an IgM antibody, ;
FACS analysis was performed on lOC1 cells and murine 238-57 ~:
ADR hybridoma cells which secrete IgG and IgM respectively. . :
Brie~ly, 1x106 cells were centrifuged at 500 x g for 3 min., washed three times with PBS and resuspended in 3 ml of PBS.
Fluorescein conjugated af~inity puri~ied F(ab)z .
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 1~-20 minutes in the ~ . :
.. . .. , . . : : . ~ ~ , . ... .. . . ..
W09l/17257 PCT/US91/02822 dark 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 ~ix the cells. The cells were incubated at 4C ;~
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 ,, ' Expressin~ Murine Hyhridoma 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.
~ ' WO 91/17257 PC~/USg1/02822 .-~ s ~ ~! 3 _ l.
It will be appreciated that the propertie~ of the antibodies examined are ef~ectively the only relevant characteristics of the corresponding hybridomas in that, ~or the purpose~ of the present invention, the hybridomas are characterized by their ability to produce particular antibodies having said properties.
Cvtotoxicity 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 Couplin~ 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 l 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 (1 x 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 (fraction~ 14-20) and these fraction~ were pooled and kept at 4C.
Celonin toxin wa~ extracted from the seeds o~ qelonium multi~lorum and puri~ied 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 addad to triethanolamine hydrochloride (TEA/HCl) bu~fer to a ~inal concentration of 60 mM TEA/HCl W~9l/17~57 PC~/US9l/0~822 and adjusted to pH 8Ø The solution was made 1 mM EDTA.
2-iminothiolane stock solution tO.5M in 0.5M TEA/HCl pH 8.0) was added to a fin~l concentration of 1 mM and the sample was incubated for 90 minutes at 4C under nitrogen gas.
Excess 2-iminothiolane rea~ent 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) ~ere 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 4C. SPDP-modified antibody 2G10 was mixed with a 5-fold molar excess of 2-iminothiolane modified gelonin. The pH of the mixture was adjusted ~o 7~0 by the addition of 0.05 M TEA/HCl buffer (pH 8.0) and the mixture was incubated for 20 hrs at 4C under nitrogen.
Iodoacetamide (O.lM in PBS) was added to a Pinal concentration of 2 mM to block any remaining free sulfhydryl groups and incubation was continued for an additional hour at 25C.
Purification of 2GlO 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 ali~uots 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 (1 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 bu~fer 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 - ~
WO91/17~57 ~r~3~ cT/us9l/n2822 fractions was determined by the dye-binding assay described previously.
The coupling mixture containing free 2GlO antibody, 2Gl0 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 2Gl0 gelonin conjugate was performed. As can be seen in Figure 9, the 2Gl0 gelonin coupling mixture (lane 3) contained free 2Gl0, free gelonin (arrows) as well as 2GlO coupled to l gelonin molecule (monomer arrow) and 2Gl0 coupled to 2 gelonin molecules (dimer, arrow ). As seen in Lane l, the final purified 2G10 :
gelonin conjugate contains mostly 2G10 coupled to l gelonin molecule and lesser amounts of 2G10 coupled to 2 gelonin molecules. The preparation was not contaminated by frea 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 în Figure lO, the 2G10 gelonin conjugate bound readily to IgM and only to a small extent to IgG coated plates. Only at the highest concentration tested (l000 ng/well) did the 2Gl0 gelonin conjugate bind significantly to the IgG-coated wells. In contrast, at a similar concentration the 2Gl0 gelonin conjugate bound extensively to the IgM coated wells.
Thus, the immunoreactivity of the 2GlO gelonin conjugate towards IgM was preserved. In addition, the 2Gl0 gelonin 3S conjugate failed to cross-react apprecia~ly with murine IgG
and therefore the selcctivity of the 2Gl0 gelonin conjugate -' ', '~
W091/172~7 PCT/US91tO2822 j~,: Z . ~ 'J `-~
was also unaltered. This data suggests that the 2GlO
gelonin conjugate should bind to IgM coating murine cells in the same manner as that of antibody 2GlO 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 invol~es immunization of murine spleen cells in vitro, i.e., in culture dishes, with ODC protein.
This teGhnique was performed by the method of Luben (Luben and Mohler, (1980) Molec. Immunol. 17:635-639) using 25 ug of purified rat liver ODC.
Briefly, ODC protein was incubated with mouse cells for 72 hours at 37C 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.
WO 91/17~57 2~ ?~ PCI'/US91102822 Tabl~ 3 ;
IN VITRO IMMUNIZATION AND HYBRIDQMA ~.
PRODUCTION SUMMARY
Production SummarY Remarks Total wells plated 48 Two 24-well plate~.
1 X 106 cells/well '~' Total wells with viable 48/48Represents a minimum ~ .
hybrid~/Total well~ plated fu~ion frequencey of ; 1:106 Total wells positive 48/4l314 of the~e were by first ELISA greater than 4-~old above control media :~
in ELISA
Wells po3itive for ODC 12/14Only the 14 most activity precipitation/ positive in EL~SA ,~, wells teQted were te3ted : Total well~ sxpanded 12/48Limited to the 12 ~ :~
for cloning most promi~ing clonss Total individual ELISA 70 As few as one positive monoclones recovered from poly-recovered ~rom 12 clonal well and as original polyclonal wells many as 18 Total monoclonals 50/70 As much as 48~ of the positive for ODC added ODC activity activity precipitation/ precipitated and as Total assaye!d little a~ 4 Total monoclone~ 70/70 secreting I~M/
total monoclones Total monoclones 0/70 ~;
aecreting IgG/total ;~
monoclones ~ , ... , : : ,. "... . . .. , . .~
W091/17257 PCT/US91tO2822 2 ~ 2~-After 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 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. ~he results of these tests are shown in Table 4.
W ~ 91/17257 ~r~ 3~ l PCT/US91/02822 Tabl~ 4 -.
IN VIVO IMMUN~ZATION AND INTR~SPECIES HY3RIDO~
PRODUCTION SUMM~Y ~;
Produ~t ion Sunu~y B~E~
Total well~ plated 1200 Numerous wells of 96-well plate~ plated with 2.5 x 105 cells/well Total wells with viable 148 Approximately 12% of : -wells hybrids plated (about 1:107 fusion freguency).
Total wells positiva 30/148 Six of these were signifi-by fir~t ELISA cantly higher than control in binding to ODC in the :
ELISA
:
Well~ cloned by limiting 30 . All positive polyclones dilution expand0d and cloned Total individual ELISA 27 Three polyclonal wells po~itive monoclones yielded no positive recovered ~rom 30 monoclones original polyclonal well~ .
Total monoclone3 secreting 27/27 No comment IgM number te~ted .~
';
Total monoclone~ ~ecreting 0/27 IgG number tested Total monoclone~ precipitating 1/5 Only one precipitated a ODC activity/number tested ~igni~icant amount o~ ODC
activity over control media ~ ' W091/17257 PCTtUS91/02822 ~ . 3~3~ 1 Twenty 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 Sortlng~ 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.
~luorescein conjugated affinity purified F(ab)2 fragment ~.
goat anti-mouse immunoglobulin IgM (Cappel) is diluted l:l00 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 S00 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 40C until sorted by flow cytometry.
For indirect staining hybridoma cells are first incubated with rat anti-mouse IgM antibody 2Gl0, washed, then stained with the fluorescein F(ab)2 fragment goat anti-mouse immunoglobulin IgM and sorted by flow cytometry.
Example 9 ;
Maqnetic 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 (2Gl0) at a concentration of approximately 0.425 mg/ml (about 0~l ml is used per l0~ calls/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 o~ 20 beads/cell. The total volume should not exceed -., wosl/172s7 PCT/US91/02822 2s!~ ~} ~ ~!' ¦
_ Sheet number 27 Itllam~onal APDIIC~On NO: PCT/
M/C:~OORCJ~fflSMS
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12301 Parklawn Drive Rockville, MD 20852 T I ,~ _ _ _ . _ _ d ~
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TU 1~11~ o - ~dll bc o~l-O Io ll~o I~UONl ~UIO~U I-l r ~5~ 1~ Oon~l ~ d Pl- Irllle-l~n ~ o '~ 01 D~n"), L 13 Tl~h or~o~ r-co~ o ~11~ nl~rndioo-l ~DDllc Uor ~h~n n~ ~10 b- Cr~d ~ IN ne 171n~r) --l ~Ç~. ~ . ' ' ~Aulron~od olnc r) O r~ ~01- 01 I~DI /l~om IU~ ollDllc~ll) b~ Ib--Illlom--UoNI tlur_u I ~
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IAulrlDrlt o omc~) ~
_ o~ ~CTII~olliU lOonu~r~ tb l) ::' .: :' : : ' ,' ' ' :: ': ' ,' :- ;. ' ' ' : . . .. , : , '' ~ ' . ' , , . :
WO91/17257 PCT/VS91~0~822 ~ 28-O. 4 ml . The cell/bead mixture is incubated for 112 hour on ice, agitating every lO 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.
What is claimed is:
;: ,. : ,:,: ,: : : : -.:: .: .. :: : . ~ . .;: :: .:;. :., ~ .. :: ,: :; . : : .. : " . .:
THEIR PRODUCTION AND US~,_AND HYBRIDOMAS FOR
PRODUCING THE SAME ~
FIELD OF_THE INVENTION ~-This invention is in the fields of immunology and monoclonal an~ibody production. More particularl~ it concerns monoclonal anti-IgM antibodies, hybridomas that produce these antibodies, immunochemicals made from those 10 antibodies, and the use of those im~unochemicals.
~ACKGROUND OF THE INVEN~IO~ -Since the report of Kohler and Milstein describing the production of monoclonal antibodies, the development of technology to produce immortalized lymphocytes capable o~
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 condi'cions.
Antibodies are endogenous proteins produced by the immune system in response to antigenic stimuli. These proteins specifically bind to antigen molecules at de~ined sites ~epitopes). Polyclonal antibodies are derived from immunization of animal~ 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) o~ cells producing a specific antibody. In contrast to polyclonal antibodies monoclonal antibodies bind to only one specific epitope on the antigen molecule. ;~
WO91~17257 PCT/US91/02~22 3 ~, l Although the technology for the generation of monoclonal antibodies has existed for some time, the current methodology is time-con~iuming, 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 hy 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 than 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 I'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 ~ia hetero-bifunctional agents. Nevelle, D.M. and Youle, R.
J., Immunol Rev (1982) 62: 75-9l; Ross, W.C.J., et al., W09lt17257 PCT/US9l/02822 -European J Biochem (1980) 104; Vitteta, E.S., et al., Immunol Rev (1982) 62: 158-183; Raso, v., et al., cancer Res (1982) 42: 457-464; Trowbridge, I.W. and Domingo, D.L., Nature tCond) (1981) 294: 171-173.
SUMMARY OF THE INVENTION
A principal aspect o~ the invention concerns rat i, monoclonal antibodies that:
(a) bind selectively to IgM sub-type antibodies;
(b) are IgGs;
(c) do not bind to IgGl or IgG2 sub-type.
The preferred embodiment of these antibodies is on~
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 khe 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 muxine 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 immunoti~xins 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 '~, .
' ~
WO91/17257 P~TIVS91/02822 cells with a cytocidally e~fective amount of one or more of the above described immunotoxins.
Other aspects of the invention are direct and indirect ~-immunoassayis 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 o~ 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 l demonstrates screening of hybridoma i~
supernatants for IgM-specific antibody.
Figure 2 demonstrates the dose-dependent specific binding of antibody 2~10.
Figure 3 demonstrates the ef~iect of increasing absorbed murine immunoglobulin content on 2Gl0 binding.
Figure 4 shows selective recognition of murine IgM by 2Gl0 in an indirect ELISA.
Figure 5 illustrates the purity of 2Gl0 antibody by SDS-PAGE.
Figure 6 characterizes the subclass of rat 2Glb antibody by Ouchterlony immunodiffusion.
Figure ~ demonstrates utilization of 2Gl0 for binding to cells expressing IgM subclass antibodies.
Figure 8 illustrates the elution profile of immunotoxin (composed of 2Gl0 coupled to gelonin) on a gel filtration matrice. . `
Figuxe 9 demonstrates the purity of 2Gl0-gelonin immunotoxin by SDS-PAGE.
Figure l0 shows the specific binding of ~r~unotoxin(2G10-ge1On1n) to rurine IgM.
' .. .
~.
wos1~172s7 ~ r~,.,3 ~ P~r/USg1/0~822 DETAILED DESCRIPTION OF THE INVENTION
In order that the invenkion herein described may be more fully understood, the following deta~lqd 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 i~ made.
As used herein with respect to the exemplified rat monoclonal anti-murine IgM antibodies, the term "~unctional 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 IgG1 or IgG2 isotype. ~;
As used herein with regard to khe 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. Pre~erably 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 IgN antibody. The rats are inoculated subcutaneously and intraperitoneally with an immunogenic amount o~ the murine IgM antibody in ~eund's adjuvant and then boosted with similar amounts of the ;~
immunogen in adjuvant. Spleens are collected from the ", ' .: ,, ... .. , ... ,. . : , . - ~ : . , . . , : . : :: . . .
W~9~/17257 PCT/US91tO2822 O' ,,., ,", ; ..., ., ~., --6-- ;
immunized 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) 18: 377-3al]. 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 IgGl, IgG2 and IgM (murine IgM antibody) as antigen. Positive clones are characterized further to ~O determine whether they meet the criteria of the invention antibodies.
Hybridomas that produce such antibodies may be grown n Yitro or in v vo using known procedures. The monoclonal antibodies may be isolated from the culture media or body 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 SeIection/Characterization The important characteristics of the monoclonal antibodies are (1) their immunoglobulln class, (2) their sel~ctivity for murine Ig~ antibody, and ~3) their usefulness in identifying and binding to murine IgM 7 producing hybridoma cells.
The selectivity and range of a given antibody is detexmined by testing it against panels of (1) IgGl, IgG2 , ,;
: `:
, 2, ~ J 1.
and IgM producing hybridoma cells and (2) IgGl, IgG2 and IgM .,.
antibodies. In selecting the claimed antibodies approximately 162 growing hybridoma cultures were initially screaned. 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 1~-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) or iminothiolane (IT) as a coupling agent. The conjugates were tested against IgM and IgG coated plates (Figure ll) to determine if specificity of the antibody is preserved after chemical coupling to the toxin.
Further details of the characterization o~ this antibody are provided in the examples below.
Immunochemicals 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 ~ragments thereof are preferred and are exemplified by gelonin, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas ~ ), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuri~es ~ordii 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 ' .~;i.. . . . , -. ... ".. . .. . . , , , ~, .. .. . ; . . . .
WO91/17257 ~.~ pCT/US91/02822 monoclonal 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 o~ imidoesters such as dimethyl adipimidate -HCl, active esters such as disuccinimidyl suberate,aldehydes such as glutaraldehyde, bis-a2ido compounds such as bis(p-azidopenzoyl) hexanediamine, bis-diazonium derivatives such as bis-(p-diamoniumbenzoyl)-ethylenediamine, diisocyanates such as tolylene 2,6-diisocyanate, and bis-active fluorine compounds such a l,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 lO nM. The formulation and mode of administration for ln vitro use are not critical. Aqueous ~ormulations 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 n 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 ~he range o~
about O.l to about lO 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 .: ';
,'. :~
`!' ' . ' ' ~ ; . , ; :
Wo91/1~257 PCT/US9~/028~2 2~ '~'~''3~
_g water, saline, Ringer's solution, dextrose solution, and 5%
human serum albumin. Nonaqueous vehicles such as fix~d 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 l mgtml to lO mg/ml.
Cytotoxic radiopharmaceuticals for eliminating IgM
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 the antibodies include moieties that may be detected directly, such as fluorochromes and radiolabels, ~s well as moieties, such as enzymes, that must be reacted or derivatized to be detected. Examples of such labels are 32p, ~25I, 3H, l4C, fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luci~eria, 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 antibodie~ may also be labeled with magnetic beads for use in magnetic sorting regimens.
The antibodies and labeled antibodies may be u~ed in a variety of cell sorting procedures to separate the IgN
producing hybridoma cells from IgG producing hybridoma cells or to eliminate the IgM producing hybridoma cells from cultures containing such cells.
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wO91/l7257 PCT/US9l/02822 Common 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 ;~
-~ith 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 lab~led or unlabeled form in suitable containers, reag~nts for tbe 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.
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 `-~:
' `, .' ,:'' ~
WO91/172~7 2~ PCT/US91/02822 dilutions to obtain a cell line that would be stable and continue producing anti-IgM antibodies.
A) Cloning by limiting dllution 58.6 cells were cultured in Iscovels medium for 3 days at 37C 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 h~macytometer. 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, a~d 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 ~or freezing of cells stocks and injections into pristane-treated nude mice for production of ascites fluid.
B) Freezing of hybrid cells When hy~rid cells plated into T75 flasks had reached 70% confluency, cells were collected by centrifugation and the pellet was rèsuspended in 0.9 ml o~ fetal bovine serum.
Immediately before freezing, the cells were transferred to `
freezing vials and O.l ml of dimethyl sulfoxide were added to each vial. The vials were stored in liquid nitrogen.
C) Ascites ~luid production Approximately 107 hybridoma cells were washed in serum-free media and injected intraperitoneally into nude mice which had received an intraperitoneal injection of 0.5 ml of pristane 7 to 14 days earlier. Ascites fluid usually formed within 1-3 weeks and waæ collected from the peritoneal cavity using a large gauge needle. Fluid was collected into tubes containing 5 ml of PBS with 20 mM EDTA. ~;
:
:
WO91/17257 PCT/U~1/02822 ~ t; . . ~ - 12-A~ter centrifugation at 2000 x g for l0 min, the supernatant was saved, made 0.1% in sodium azide, and stored at 4C or frozen at -20C in small ali~uots. This fluid provided a rich source of monoclonal antibody (approximately 5-l0 mg/ml).
Example 2 Selection and Assav of Hvbridoma Cells Producin~ Rat Anti-mouse IqM Antibodies ~ybridoma colonies which grew to a density of approximately 500-lOO0 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 ~rom these colonies was as~ayed 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). l00 mg of l 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 4C in a humidified chamber. The wells were then washed three times with phosphate-buffered saline containing 0.2% Tween-20 ;
(PBS-~ween). After washing and removal of all traces of liquid in the wells by tapping lightly onto paper towels, l00 ug o~ hybridoma supernatant was added to the IgM-coated wells ~nd incubated a~ room temperature for 2 hours. Plates were again washed with PBS-Tween, and incubated for one hour at room temperature with l00 ul of a l:l000 dilution of peroxidase conjugated goat anti-rat IgG in PBS-Tween. After the wells were washed again as described above, they were reacted with l00 ul o~ 1 mM ABTS ~ ~`
(2,2-azino-di(3-ethylbenzthiazoline sulphonic acid) in 0.l M
sodium citrate buffer pH 4.2, containing 0.03% hydrogen ~;
peroxide) for 20-60 min at 37C. Optical density was measured at 405 nm on a NicroElisa Reader.
.. : ~ . .. :; .. : . . , . , , , ,,. .. , : ~ . . .. .
WO91/17257 ~ PCT/US91/02822 In 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-~appa protein coated plates was tested. Hybridomas IC2 and 2GlO 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.
~hat the other 7 antibodies tested also bound to murine IgM
is indicated by the results shown on Figure l. As can be seen in Figure lA and lB, nine different hybridomas were tested against either IgM-kappa or IgM-lambda coated plates.
A sta~dard ELISA assay was performed to measure rat immunization bound to each plate. All antibodies were found to bind to both IgM-k and IgM-l coated wells. Among the highest binding antibodies, lC2 and 2GlO were chosen for further study. Because of its growth and antibody production characteristics, antibody 2GlO 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 TgM
antibody of greater than or equal to 0.3 O.D. were saved. ~ ~`
The specificity of the rat monoclonal 2GlO antibody was tested by ELISA under a variety of conditions. To determine the selective recognition of IgM vs. IgG by 2GlO lOOng of mouse IgM-lambda protein or IgG3-lambda protein was absorbed onto microtiter plates (lO0 ul) and incubated with increasing concentrations of 2GlO antibody. As shown in Figure 2, in the antibody dose range of 2-lO00 ng, the 2GlO
hound to IgM-coated microtiter plates to a greater extent than that achieved in IgG-coated plates. At 2GlO antibody doses of 50 ng and below, no binding was measureable on IgG-coated plates whereas IgM-coated wells were recognized by 2GlO.
WO 91/17257 PcI`/US91/o2822 J`'J ~' -14-The effect of increasing immunoglobulin coating concentration on microtiter plates and recognition by antibody 2G10 was also examin~d. 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 IqM-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 indixect 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, ;~, incxeasing concentrations of ra~ 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 ko its respective antigen (closed circles), but was unable to detect IgG (open circles) under the same conditions, although the presence of IgG1 could be easily detected with antibodies reactive with all mouse immunoglobulin subtypes. These results demonstrate the selective recognition of mouse IgM subclass immunoglobulins by ~G10 rat monoclonal antibody. The rat monoclonal ;~
antibody was also able to recognize IgM antibody at picogram doses, demonstating its high a~finity of murine IgM
antibody.
Example 3 Characterization of Rat Anti-Mouse IgM Antibodies Mouse immunoglobulin of various subtypes (IgM-kappa and lambda, IgG1, 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 .
WO91/17257 ~ PCT~US9l/02822 positi~e 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 2GlO rat monoclonal antibody A representative rat anti-mouse IgM monoclonal antibody, designated 2GlO, was chosen for further characterization. As was shown in Example 3, the 2GlO
antibody was positive for IgM kappa and lambda. The antibody was purified by centrifugation and ammonium sulfate fractionation.
~he representative hybridoma cell line designated 2GlO
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. ~he deposits are available pursuant to the patent laws and regulations of the United States and of those countries foreign to the United S~ates 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.
2GlO 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 sul~ate solution. The sample was stirxed for 30 minutes at 4C and then centrifuged at 20,000 x g for 30 minutes~ The pellet was rssuspended 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 lO0 volumes of PBS. Aliquots of the solution was used for determination of protéin content (by optica1 density at 280nm), purity (by .
W~91~17257 PCT/US91/02822 ~ 16-SDS-PAGE) and binding speci~icity (by ELISA). ~he remaining antibody solution was frozen at -20C until needed.
IgM antibodies were purified by ammonium sulfate precipitation and gel ~iltration on a 2.6 x 40 cm column o~
chromatographic resin containing agarose, dextran andtor acrylamide eluting with PBS/0.01~ sodium azide at room temperature at a flow rate o~ 1 ml/min.
The subclass of 2G10 rat monoclonal antibody was determined by the method of Ouchkerlony (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.).
~he subclass of antibody ~G10 is important in evaluating how to purify this molecule. To perform subclass analysis, an Ouchterlony immunodiffuæion 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-l 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, '~
' : -~:
' .. :......... .. . .
W09tJ1725~ PCT/US91tO2822 ~r~' there was no binding of this rat antibody to murine IgM
coating the wells.
Table 1 Evaluation of commercially available rat antibodies 5against murine IgM
ELISA reactivity aaainst murine_Ia oncentration ~ gamma-k qamma-l lOOO ng 0.058 00008 0.037 500 ng 0.034 0.004 0.031 :~ -~
10 100 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 15 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 20 SDS PAGE.
Example 5 Bindina of Rat Anti-Mouse IqM to IaM Producinq Cells In order to demonstrate that the 2G10 rat anti-mouse :
IgM antibody bound not only to purified IgM antibody coating 25 a 96 well plate but also to cells producing an IgM antibody, ;
FACS analysis was performed on lOC1 cells and murine 238-57 ~:
ADR hybridoma cells which secrete IgG and IgM respectively. . :
Brie~ly, 1x106 cells were centrifuged at 500 x g for 3 min., washed three times with PBS and resuspended in 3 ml of PBS.
Fluorescein conjugated af~inity puri~ied F(ab)z .
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 1~-20 minutes in the ~ . :
.. . .. , . . : : . ~ ~ , . ... .. . . ..
W09l/17257 PCT/US91/02822 dark 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 ~ix the cells. The cells were incubated at 4C ;~
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 ,, ' Expressin~ Murine Hyhridoma 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.
~ ' WO 91/17257 PC~/USg1/02822 .-~ s ~ ~! 3 _ l.
It will be appreciated that the propertie~ of the antibodies examined are ef~ectively the only relevant characteristics of the corresponding hybridomas in that, ~or the purpose~ of the present invention, the hybridomas are characterized by their ability to produce particular antibodies having said properties.
Cvtotoxicity 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 Couplin~ 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 l 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 (1 x 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 (fraction~ 14-20) and these fraction~ were pooled and kept at 4C.
Celonin toxin wa~ extracted from the seeds o~ qelonium multi~lorum and puri~ied 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 addad to triethanolamine hydrochloride (TEA/HCl) bu~fer to a ~inal concentration of 60 mM TEA/HCl W~9l/17~57 PC~/US9l/0~822 and adjusted to pH 8Ø The solution was made 1 mM EDTA.
2-iminothiolane stock solution tO.5M in 0.5M TEA/HCl pH 8.0) was added to a fin~l concentration of 1 mM and the sample was incubated for 90 minutes at 4C under nitrogen gas.
Excess 2-iminothiolane rea~ent 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) ~ere 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 4C. SPDP-modified antibody 2G10 was mixed with a 5-fold molar excess of 2-iminothiolane modified gelonin. The pH of the mixture was adjusted ~o 7~0 by the addition of 0.05 M TEA/HCl buffer (pH 8.0) and the mixture was incubated for 20 hrs at 4C under nitrogen.
Iodoacetamide (O.lM in PBS) was added to a Pinal concentration of 2 mM to block any remaining free sulfhydryl groups and incubation was continued for an additional hour at 25C.
Purification of 2GlO 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 ali~uots 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 (1 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 bu~fer 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 - ~
WO91/17~57 ~r~3~ cT/us9l/n2822 fractions was determined by the dye-binding assay described previously.
The coupling mixture containing free 2GlO antibody, 2Gl0 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 2Gl0 gelonin conjugate was performed. As can be seen in Figure 9, the 2Gl0 gelonin coupling mixture (lane 3) contained free 2Gl0, free gelonin (arrows) as well as 2GlO coupled to l gelonin molecule (monomer arrow) and 2Gl0 coupled to 2 gelonin molecules (dimer, arrow ). As seen in Lane l, the final purified 2G10 :
gelonin conjugate contains mostly 2G10 coupled to l gelonin molecule and lesser amounts of 2G10 coupled to 2 gelonin molecules. The preparation was not contaminated by frea 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 în Figure lO, the 2G10 gelonin conjugate bound readily to IgM and only to a small extent to IgG coated plates. Only at the highest concentration tested (l000 ng/well) did the 2Gl0 gelonin conjugate bind significantly to the IgG-coated wells. In contrast, at a similar concentration the 2Gl0 gelonin conjugate bound extensively to the IgM coated wells.
Thus, the immunoreactivity of the 2GlO gelonin conjugate towards IgM was preserved. In addition, the 2Gl0 gelonin 3S conjugate failed to cross-react apprecia~ly with murine IgG
and therefore the selcctivity of the 2Gl0 gelonin conjugate -' ', '~
W091/172~7 PCT/US91tO2822 j~,: Z . ~ 'J `-~
was also unaltered. This data suggests that the 2GlO
gelonin conjugate should bind to IgM coating murine cells in the same manner as that of antibody 2GlO 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 invol~es immunization of murine spleen cells in vitro, i.e., in culture dishes, with ODC protein.
This teGhnique was performed by the method of Luben (Luben and Mohler, (1980) Molec. Immunol. 17:635-639) using 25 ug of purified rat liver ODC.
Briefly, ODC protein was incubated with mouse cells for 72 hours at 37C 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.
WO 91/17~57 2~ ?~ PCI'/US91102822 Tabl~ 3 ;
IN VITRO IMMUNIZATION AND HYBRIDQMA ~.
PRODUCTION SUMMARY
Production SummarY Remarks Total wells plated 48 Two 24-well plate~.
1 X 106 cells/well '~' Total wells with viable 48/48Represents a minimum ~ .
hybrid~/Total well~ plated fu~ion frequencey of ; 1:106 Total wells positive 48/4l314 of the~e were by first ELISA greater than 4-~old above control media :~
in ELISA
Wells po3itive for ODC 12/14Only the 14 most activity precipitation/ positive in EL~SA ,~, wells teQted were te3ted : Total well~ sxpanded 12/48Limited to the 12 ~ :~
for cloning most promi~ing clonss Total individual ELISA 70 As few as one positive monoclones recovered from poly-recovered ~rom 12 clonal well and as original polyclonal wells many as 18 Total monoclonals 50/70 As much as 48~ of the positive for ODC added ODC activity activity precipitation/ precipitated and as Total assaye!d little a~ 4 Total monoclone~ 70/70 secreting I~M/
total monoclones Total monoclones 0/70 ~;
aecreting IgG/total ;~
monoclones ~ , ... , : : ,. "... . . .. , . .~
W091/17257 PCT/US91tO2822 2 ~ 2~-After 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 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. ~he results of these tests are shown in Table 4.
W ~ 91/17257 ~r~ 3~ l PCT/US91/02822 Tabl~ 4 -.
IN VIVO IMMUN~ZATION AND INTR~SPECIES HY3RIDO~
PRODUCTION SUMM~Y ~;
Produ~t ion Sunu~y B~E~
Total well~ plated 1200 Numerous wells of 96-well plate~ plated with 2.5 x 105 cells/well Total wells with viable 148 Approximately 12% of : -wells hybrids plated (about 1:107 fusion freguency).
Total wells positiva 30/148 Six of these were signifi-by fir~t ELISA cantly higher than control in binding to ODC in the :
ELISA
:
Well~ cloned by limiting 30 . All positive polyclones dilution expand0d and cloned Total individual ELISA 27 Three polyclonal wells po~itive monoclones yielded no positive recovered ~rom 30 monoclones original polyclonal well~ .
Total monoclone3 secreting 27/27 No comment IgM number te~ted .~
';
Total monoclone~ ~ecreting 0/27 IgG number tested Total monoclone~ precipitating 1/5 Only one precipitated a ODC activity/number tested ~igni~icant amount o~ ODC
activity over control media ~ ' W091/17257 PCTtUS91/02822 ~ . 3~3~ 1 Twenty 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 Sortlng~ 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.
~luorescein conjugated affinity purified F(ab)2 fragment ~.
goat anti-mouse immunoglobulin IgM (Cappel) is diluted l:l00 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 S00 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 40C until sorted by flow cytometry.
For indirect staining hybridoma cells are first incubated with rat anti-mouse IgM antibody 2Gl0, washed, then stained with the fluorescein F(ab)2 fragment goat anti-mouse immunoglobulin IgM and sorted by flow cytometry.
Example 9 ;
Maqnetic 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 (2Gl0) at a concentration of approximately 0.425 mg/ml (about 0~l ml is used per l0~ calls/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 o~ 20 beads/cell. The total volume should not exceed -., wosl/172s7 PCT/US91/02822 2s!~ ~} ~ ~!' ¦
_ Sheet number 27 Itllam~onal APDIIC~On NO: PCT/
M/C:~OORCJ~fflSMS
, ~
A.
~ ~O~
_ d ~
American T~pe Cul~ure Collection ~ d ~
12301 Parklawn Drive Rockville, MD 20852 T I ,~ _ _ _ . _ _ d ~
23 April 1990 ¦ ~B 10436 . ~T~ ~a~ bn~ 1. Tbl~ Irht~ll*t 1~ t ~ ~ O
, ~ .
~u~un~n--~o~w~lo~ I~DICAT OU--A~t U~D~ I (111~1~- ~ ~ ~ ~ ~ ~1 .,_ ,~
. .
..... ~
D~ IWRUI--~UU--0~ lClUrlOI~ Inr~ 11 not ~ lk~) - - .
TU 1~11~ o - ~dll bc o~l-O Io ll~o I~UONl ~UIO~U I-l r ~5~ 1~ Oon~l ~ d Pl- Irllle-l~n ~ o '~ 01 D~n"), L 13 Tl~h or~o~ r-co~ o ~11~ nl~rndioo-l ~DDllc Uor ~h~n n~ ~10 b- Cr~d ~ IN ne 171n~r) --l ~Ç~. ~ . ' ' ~Aulron~od olnc r) O r~ ~01- 01 I~DI /l~om IU~ ollDllc~ll) b~ Ib--Illlom--UoNI tlur_u I ~
. ~
~ . =~ .
IAulrlDrlt o omc~) ~
_ o~ ~CTII~olliU lOonu~r~ tb l) ::' .: :' : : ' ,' ' ' :: ': ' ,' :- ;. ' ' ' : . . .. , : , '' ~ ' . ' , , . :
WO91/17257 PCT/VS91~0~822 ~ 28-O. 4 ml . The cell/bead mixture is incubated for 112 hour on ice, agitating every lO 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.
What is claimed is:
;: ,. : ,:,: ,: : : : -.:: .: .. :: : . ~ . .;: :: .:;. :., ~ .. :: ,: :; . : : .. : " . .:
Claims (28)
[received by the International Bureau on 10 October 1991 (10.10.91);
original claim 7 cancelled; claims 1-6 amended; claims 8-12 amended and renumbered as claims 7-11; new claims 12-28 added (4 pages)]
1. An anti-IgM antibody conjugate comprising:
a monoclonal antibody which binds selectively to IgM antibody, does not bind to IgG1 or IgG2 antibody, and has a G isotype; and a cytotoxic moiety conjugated to said monoclonal antibody.
a monoclonal antibody which binds selectively to IgM antibody, does not bind to IgG1 or IgG2 antibody, and has a G isotype; and a cytotoxic moiety conjugated to said monoclonal antibody.
2. An anti-IgM antibody conjugate comprising:
a monoclonal antibody which binds selectively to murine IgM antibody, does not bind to IgG1 or IgG2 antibody, and has a G isotype; and a cytotoxic moiety conjugated to said monoclonal antibody.
a monoclonal antibody which binds selectively to murine IgM antibody, does not bind to IgG1 or IgG2 antibody, and has a G isotype; and a cytotoxic moiety conjugated to said monoclonal antibody.
3. The conjugate of Claim 1, wherein said monoclonal antibody binds to hybridoma cells producing an IgM antibody.
4. The conjugate of Claim 1, wherein said monoclonal antibody binds to hybridoma cells producing a murine IgM
antibody.
antibody.
5. The conjugate of Claim 1, wherein said monoclonal antibody is produced by one of the following hybridomas:
(a) 2G10;
(b) 1C2; or is a monoclonal antibody which is functionally equivalent to any one of the aforesaid antibodies.
(a) 2G10;
(b) 1C2; or is a monoclonal antibody which is functionally equivalent to any one of the aforesaid antibodies.
6. The conjugate of Claim 1, wherein said monoclonal antibody is produced by a rat X rat hybridoma or progeny of said hybridoma.
7. An immunotoxin which is a conjugate of a cytotoxic moiety and a monoclonal antibody which binds selectively to IgM antibody, does not bind to IgG1 or IgG2 antibody, and has a G isotype.
8. A method of killing murine IgM antibody producing cells comprising contacting said cells with a cytocidally effective amount of an immunotoxin as defined in Claim 8.
9. The conjugate of Claim 1, wherein said monoclonal antibody is labeled with a detectable label.
10. A method for collecting hybridoma producing IgG
isotype monoclonal antibodies, comprising:
treating a hybrid cell population with a monoclonal antibody which has a g isotype and binds selectively to IgM antibody but does not bind to IgG
or IgG2 antibody;
subjecting said resulting immunocomplexed cells to sorting; and collecting the cells which have not complexed with said antibodies.
isotype monoclonal antibodies, comprising:
treating a hybrid cell population with a monoclonal antibody which has a g isotype and binds selectively to IgM antibody but does not bind to IgG
or IgG2 antibody;
subjecting said resulting immunocomplexed cells to sorting; and collecting the cells which have not complexed with said antibodies.
11. A method of making an immunotoxin, comprising:
a monoclonal antibody which binds selectively to IgM antibody, does not bind to IgG1 or IgG2 antibody, and has a G isotype; and a cytotoxic moiety conjugated to said monoclonal antibody.
a monoclonal antibody which binds selectively to IgM antibody, does not bind to IgG1 or IgG2 antibody, and has a G isotype; and a cytotoxic moiety conjugated to said monoclonal antibody.
12. The conjugate of Claim 2, wherein said monoclonal antibody binds to hybridoma cells producing an anti-IgM
antibody.
antibody.
13. The conjugate of Claim 2, wherein said monoclonal antibody binds to hybridoma cells producing an anti-mouse IgM antibody.
14. The conjugate of Claim 2, wherein said monoclonal antibody is labeled with a detectable label.
15. The conjugate of Claim 2, wherein said monoclonal antibody is either produced by 2G10 or 1C2 hybridomas or is a functional equivalent to a monoclonal antibody produced by 2G10 or 1C2 hybridomas.
16. The conjugate of Claim 2, wherein said monoclonal antibody is produced by a rat X rat hybridomas or progeny of said hybridoma.
17. The conjugate of Claim 3, wherein said monoclonal antibody is labeled with a detectable label.
18. The conjugate of Claim 3, wherein said monoclonal antibody is either produced by 2G10 or 1C2 hybridomas or is a functional equivalent to a monoclonal antibody produced by 2G10 or 1C2 hybridomas.
19. The conjugate of Claim 3, wherein said monoclonal antibody is produced by a rat X rat hybridomas or progeny of said hybridoma.
20. The conjugate of Claim 4, wherein said monoclonal antibody is produced by a rat X rat hybridomas or progeny of said hybridoma.
21. The conjugate of Claim 5, wherein said monoclonal antibody is produced by a rat X rat hybridomas or progeny of said hybridoma.
22. An immunotoxin which is a conjugate of a cytotoxic moiety and a monoclonal antibody which binds selectively to murine IgM antibody, does not bind to IgG1 or IgG2 antibody, and has a G isotype.
23. An immunotoxin which is a conjugate of a cytotoxic moiety and an anti-IgM monoclonal antibody which binds selectively to antibody, does not bind to IgG1 or IgG2 antibody, and has a G isotype.
24. A method for collecting hybridoma producing IgG
isotype monoclonal antibodies, comprising:
treating a hybrid cell population with a monoclonal antibody which has a g isotype and binds selectively to IgM antibody but does not bind to IgG1 or IgG2 antibody;
subjecting said resulting immunocomplexed cells to sorting;
and collecting the cells which have not complexed with said antibodies.
isotype monoclonal antibodies, comprising:
treating a hybrid cell population with a monoclonal antibody which has a g isotype and binds selectively to IgM antibody but does not bind to IgG1 or IgG2 antibody;
subjecting said resulting immunocomplexed cells to sorting;
and collecting the cells which have not complexed with said antibodies.
25. The method of Claim 24, wherein said immunocomplexed cells are sorted by flow cytometry.
26. The method of Claim 24, wherein said immunocomplexed cells are sorted by magnetic cell separation.
27. The method of Claim 10, wherein said immunocomplexed cells are sorted by flow cytometry.
28. The method of Claim 10, wherein said immunocomplexed cells are sorted by magnetic cell separation.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US51597490A | 1990-04-27 | 1990-04-27 | |
| US515,974 | 1990-04-27 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2079901A1 true CA2079901A1 (en) | 1991-10-28 |
Family
ID=24053580
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002079901A Abandoned CA2079901A1 (en) | 1990-04-27 | 1991-04-24 | Monoclonal anti-igm antibodies, their production and use, and hybridomas for producing the same |
Country Status (12)
| Country | Link |
|---|---|
| EP (1) | EP0538267A4 (en) |
| JP (1) | JPH06504424A (en) |
| CN (1) | CN1057072A (en) |
| CA (1) | CA2079901A1 (en) |
| IE (1) | IE911172A1 (en) |
| IL (1) | IL97827A0 (en) |
| NZ (1) | NZ237784A (en) |
| PT (1) | PT97482B (en) |
| RU (1) | RU2105062C1 (en) |
| TW (1) | TW349995B (en) |
| WO (1) | WO1991017257A1 (en) |
| ZA (1) | ZA912695B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MY171554A (en) | 2008-09-05 | 2019-10-18 | Sekisui Medical Co Ltd | Monoclonal antibody and immunoassay using the same |
| CN108384761B (en) * | 2018-03-30 | 2020-08-11 | 四川迈克生物新材料技术有限公司 | Anti-human IgM monoclonal antibody, hybridoma cell strain and application thereof |
| WO2023088443A1 (en) * | 2021-11-20 | 2023-05-25 | 东莞市朋志生物科技有限公司 | Anti-human igm antibody and preparation method therefor and use thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4792447A (en) * | 1981-07-23 | 1988-12-20 | Board Of Regents, The University Of Texas System | Anti-immunoglobulin toxin conjugates useful in the treatment of B cell tumors |
| AU9016591A (en) * | 1990-10-25 | 1992-05-26 | Tanox Biosystems, Inc. | Glycoproteins associated with membrane-bound immunoglobulins as antibody targets on B cells |
-
1991
- 1991-04-08 IE IE117291A patent/IE911172A1/en unknown
- 1991-04-10 NZ NZ237784A patent/NZ237784A/en unknown
- 1991-04-11 ZA ZA912695A patent/ZA912695B/en unknown
- 1991-04-11 IL IL97827A patent/IL97827A0/en unknown
- 1991-04-17 TW TW080103007A patent/TW349995B/en active
- 1991-04-24 WO PCT/US1991/002822 patent/WO1991017257A1/en not_active Application Discontinuation
- 1991-04-24 JP JP3509144A patent/JPH06504424A/en active Pending
- 1991-04-24 EP EP19910909436 patent/EP0538267A4/en not_active Withdrawn
- 1991-04-24 RU RU92016351A patent/RU2105062C1/en active
- 1991-04-24 CA CA002079901A patent/CA2079901A1/en not_active Abandoned
- 1991-04-26 PT PT97482A patent/PT97482B/en not_active IP Right Cessation
- 1991-04-27 CN CN91102913A patent/CN1057072A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| EP0538267A1 (en) | 1993-04-28 |
| JPH06504424A (en) | 1994-05-26 |
| CN1057072A (en) | 1991-12-18 |
| PT97482A (en) | 1992-01-31 |
| ZA912695B (en) | 1993-02-24 |
| EP0538267A4 (en) | 1994-06-01 |
| IL97827A0 (en) | 1992-06-21 |
| WO1991017257A1 (en) | 1991-11-14 |
| RU2105062C1 (en) | 1998-02-20 |
| NZ237784A (en) | 1992-09-25 |
| AU7866491A (en) | 1991-11-27 |
| PT97482B (en) | 1998-08-31 |
| AU650659B2 (en) | 1994-06-30 |
| IE911172A1 (en) | 1991-11-06 |
| TW349995B (en) | 1999-01-11 |
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