RU2105062C1 - ANTI-iGM-ANTIBODY-BASE CONJUGATE (VARIANTS) AND METHOD OF DECREASE OF IGM-ANTIBODY SECRETION BY LYMPHOCYTES (VARIANTS) - Google Patents

Abstract

FIELD: biotechnology, immunology. SUBSTANCE: method of decrease of IgM-antibody secretion from lymphocytes involves contact of lymphocytes with cytocidal effective amount of conjugate based on anti-IgM-antibody. Conjugate has cytotoxic agent gelonin bound with monoclonal antibody which interacts selectively with IgM-antibody or hybridoma cells producing IgM-antibody but does not interact with IgG1- and IgG2-antibody and relates to G-isotype. EFFECT: improved method of secretion decrease. 8 cl, 4 tbl, 7 dwg

Description

 The invention relates to the field of immunology, in particular to the production and use of monoclonal antibodies. More specifically, it relates to monoclonal anti-IgM antibodies, hybridomas that produce these antibodies, immunochemical agents derived from these antibodies, and their use.

 Since the report of Kohler and Milstein, which described the preparation of monoclonal antibodies, the development of technology for producing lymphocytes capable of producing antibodies of a specific specificity in advance has been aimed at the development of both applied and fundamental scientific research.

 Antibodies are endogenous proteins produced by the immune system in response to antigenic stimulation. These proteins specifically bind to antigen molecules in specific areas (epitopes). Polyclonal antibodies are obtained by immunizing animals with antigens, such antibodies bind to the antigen molecule in several areas (epitopes). Monoclonal antibodies, on the other hand, are a very definite set of antibodies that are obtained from a single clone (monoclone) of cells that produce a specific antibody. Unlike polyclonal antibodies, monoclonal antibodies bind to only one specific epitope on an antigen molecule.

 Although the technology for producing monoclonal antibodies is quite well developed, modern methods still remain very time-consuming, laborious and often lead to the production of antibodies, which, although they are specific for the target antigen, have relatively low affinity for it, and thus have restrictions on use.

 Among the difficulties encountered in obtaining effective and clinically useful monoclonal antibodies, the most important is the abundance of IgM subtype antibodies obtained from hybridomas produced using standard in vivo or in vitro immunization procedures. IgM subtype antibodies generally have low affinity, are difficult to purify, and often contain many antibodies produced by hybridomas. In addition, in mixed cultures of IgM and IgG secreting hybridoma cells, IgM secreting cells often outgrow IgG secreting hybrid cells.

 Part of the time-consuming procedure for obtaining hybridomas is the removal of hybridoma cells producing IgM obtained after cell fusion. This is generally done by cloning cells using limited dilution, growing individual cells in colonies, and testing each colony separately to determine which colonies produce IgG subtype antibodies. Typically, IgG producing hybridoma cells are then subjected to a subsequent analysis in order to determine the antigenic specificity of the antibodies produced.

 There are reports of antibodies that are directed against epitopes on an IgM antibody, but all of these antibodies tested to date have also been shown to be chemically active with IgG subtype antibodies.

 The applicant, as well as other authors have proposed various cytotoxic agents that bind to antibodies with the formation of so-called immunotoxins. Recently, the interest of researchers has been focused mainly on immunotoxins, in which monoclonal antibodies are conjugated to enzymatically active sites (A-chains) of toxins of bacterial or plant nature through hetero-bifunctional agents (Nevelle, DM and Youle, RJ, Immunol Rev (1982) vol. 62, pp. 75-91, Ross, WCJ et al. European J. Biochem. (1980), pp. 104, Vitteta, ES et al. Immunol. Rev. (1982), vol. 62, pp. 158 -183, Raso, V. et al. Cancer. Res. (1982), v. 42, p. 457-464, Trowbridge, IW and Domingo DL, Nature (London) (1981), v. 294, p. 171 -173.

The present invention relates to the production and use of rat monoclonal antibodies, which:
(a) selectively bind to IgM subtype antibodies;
(b) are IgG;
(c) do not bind to subtypes of IgG ₁ or IgG ₂ .

 (A preferred embodiment of these 2G10 antibodies and its functional equivalents).

More specifically, the present invention relates to conjugation immunotoxins.
(a) the above monoclonal antibodies and
(b) a cytotoxic component or magnetic beads.

 In accordance with another aspect, the present invention relates to a method for inhibiting lymphocyte secretion of IgM antibodies by contacting these cells with a cytocidally effective amount of one or more of the above immunotoxins.

 Additional aspects of the present invention include direct or indirect immunoassays directed to the detection of IgM antibody producing cells or to the detection of an IgM isotype antibody. These assays include incubating cells with monoclonal antibodies or its labeled derivatives. If labeled derivatives are used, the presence of labeled binary immune complexes on the cells is read directly. If an unlabeled antibody is used, the cells are further incubated with a labeled antibody against a monoclonal antibody and the presence of labeled tertiary complexes on the cells is detected.

 FIG. 1 and 2 show testing of hybridoma supernatants for an IgM-specific antibody.

 FIG. 3 shows the specific binding of 2G10 antibody in a dose-dependent manner.

 FIG. 4 shows the effect of increasing the content of absorbed mouse immunoglobulin upon 2G10 binding.

 In FIG. Figure 5 shows the selective recognition of mouse IgM by direct enzyme-linked immunosorbent assay (ELISA) using 2G10.

 In FIG. Figure 6 shows the purity of the 2G10 antibody by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PACE).

 FIG. 7 characterizes a subclass of rat 2G10 antibody by Ouchterlony immunodiffusion.

 FIG. 8-11 demonstrate the use of 2G10 for binding to cells expressing antibodies of the IgM subclass.

 FIG. 12 illustrates an elution profile of an immunotoxin (obtained from binding of 2G10 to gelonin) on a gel filtration matrix.

 FIG. 13 shows the purity of the 2G10-gelonin immunotoxin using SDS-PAGE.

 In FIG. 14 shows the specific binding of immunotoxin (2G10-gelonin) to mouse IgM.

 In FIG. Figure 15 shows the effect of conjugate treatment on IgM secretion by a primary lymphocyte culture after 24 hours I.

 In FIG. Figure 16 shows the effect of conjugate treatment on IgM secretion by a primary lymphocyte culture after 72 hours I.

 In FIG. 17 shows the effect of conjugate treatment on IgM secretion by a primary lymphocyte culture after 96 hours I.

 In FIG. Figure 18 shows the effect of conjugate treatment on IgM secretion by a primary lymphocyte culture after 24 hours II.

 In FIG. 19 shows the effect of conjugate treatment on IgM secretion by a primary lymphocyte culture after 48 hours II.

 In FIG. 20 shows the effect of conjugate treatment on IgM secretion by a primary lymphocyte culture after 72 hours II.

 In FIG. Figure 21 shows the effect of a 2-hour conjugate treatment on IgM secretion by a primary lymphocyte culture after 24 hours III.

 In FIG. Figure 22 shows the effect of a 2-hour treatment on IgM secretion by a primary lymphocyte culture after 48 hours III.

 In FIG. 23 shows the effect of a 2-hour treatment on IgM secretion by a primary lymphocyte culture after 72 hours III.

 In FIG. 24 shows the effect of a 2-hour treatment on IgM secretion by a primary lymphocyte culture after 96 hours III.

 In FIG. 25 shows a comparison of IgM and IgG secretion in the conjugate and the processed AB / gel / antibody / gelonin fusions.

 In FIG. Figure 26 shows the detection of IgM in mouse serum I previously immunized with KLH.

 In FIG. 27 shows detection of IgM in mouse serum II pre-immunized with KLH.

 As used herein, the term "monoclonal antibody" refers to an antibody composition represented by a homogeneous population.

The term “functional equivalent” with respect to, for example, rat monoclonal anti-mouse IgM antibodies, refers to a monoclonal antibody that: (a) crossblocks a rat monoclonal antibody, (b) selectively binds to a murine IgM antibody, (c) has a q isotype, and (d) does not bind to either the IgG ₁ isotype or the IgG ₂ isotype.

 The term “offspring”, as used herein relative to hybridomas producing the monoclonal antibodies of the present invention, is intended to “encompass” all derivatives and products of the parent hybridoma.

 The present invention can be used to produce antibodies that will bind to any kind of IgM antibodies. The description of the present invention discloses the production of a hybridoma cell line that is stable and continues to produce an anti-IgM antibody directed to an immunizing species. In a preferred embodiment, the anti-IgM monoclonal antibody of the invention is directed to murine or human IgM.

Monoclonal Antibody Production
The fusion partners providing the hybridoma of the present invention are obtained by immunizing rats with a mouse IgM antibody. Rats were inoculated subcutaneously and intraperitoneally with an immunogenic amount of mouse IgM antibody in Freund's adjuvant, which was then increased by administering a similar amount of immunogen in the adjuvant. A few days after the last activation of immunization, the spleens of the immunized rats were removed and a cell suspension was prepared for use in the fusion.

 Hybridomas were obtained from splenocytes and rat tumor cells using the hybridization technique of somatic cells by Kohler B. and Milstein C. (Nature (1975), v. 256, pp. 495-497) modified by Buck et al. (In vitro, (1982) , t. 18, pp. 377-381). For hybridization, available rat myeloma lines, such as UV2 / 0 and ultrasound-Ag 1.2.3, can be used.

Basically, these techniques include the fusion of tumor cells and splenocytes using a substance that promotes cell fusion, such as polyethylene glycol. After fusion, the cells were separated from the medium and grown in selective medium, such as, for example, NAT-medium, in order to remove non-hybridized parent cells. Hybridomas were propagated, if necessary, and supernatants were analyzed for anti-mouse IgM activity using known immunoassay techniques (e.g., radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay) using the immunizing agent IgG ₁ , IgG ₂ and IgM / mouse IgM- antibody / as antigen. Positive clones were further characterized in order to determine whether they met the criteria of the antibodies of the present invention.

 Hybridomas that produce such antibodies can be grown in vitro or in vivo using known techniques. Monoclonal antibodies can be isolated from the culture medium (or body fluids, as the case may be) using known immunoglobulin purification techniques such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography and ultrafiltration, if necessary.

Monoclonal Antibody Selection / Characterization
Important characteristics of monoclonal antibodies are (1) the class of immunoglobulin, (2) selectivity for mouse IgM antibodies, and (3) applicability in the identification and binding of hybridoma producing mouse IgM to cells.

The selectivity and spectrum of this antibody was determined by testing it with (1) IgG ₁ , IgG ₂ and IgM-producing hybridoma cells and (2) IgG ₁ , IgG ₂ and IgM antibodies. In the selection of the proposed antibodies, approximately 162 growing hybridoma cultures were initially screened. Nine clones interacted with a murine IgM antibody, but not with IgG. One of these clones was selected for further characterization.

 Antibodies exhibiting acceptable selectivity and spectra were conjugated to gelonin using N-succinimidyl-3- (2-pyridylthio) propionate) SPD) or iminothiolane (IT) as a binding agent. Conjugates were tested against IgM and IgG coated plates (FIG. 2) to determine if antibody specificity was maintained after chemical mating with the toxin.

 Further details of the characterization of this antibody will be given in the measurements below.

Immunochemical agents
Immunochemical derivatives of monoclonal antibodies of the present invention, which are of particular importance, are immunotoxins / conjugates of the antibody and the cytotoxic component and labeled, for example, radiolabeled, enzyme labeled, magnetically labeled or phosphor labeled, derivatives in which the label is a means for identification and / or sorting immune complexes, including labeled antibody.

 The cytotoxic component of an immunotoxin can be either a cytotoxic preparation, or an enzymatically active toxin of a bacterial or plant nature, or an enzymatically active fragment (A chain) of such a toxin. Enzymatically active toxins and fragments thereof are preferred, examples include gelonin, diphtheria toxin A chain, non-binding active diphtheria toxin fragments, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, arbin A chain, modcin A chain , alpha-sarcinol, Aleurites fordii proteins, diantin proteins, Phytoiacca americana proteins (PAPI, PAPII and PAP-S), Momordica Charantia inhibitor, curcins, crotin, Saponaria officinalis inhibitor, mitocellin, restricttocin, phenomycin and enomycin. Gelonin is most preferred. Monoclonal antibody conjugates and such cytotoxic moieties can be prepared using a wide variety of coupling agents for the preparation of bifunctional proteins. Examples of such reagents are LDPE, IT, bifunctional imidoester derivatives, such as dimethyl adipimidate-HC1, active esters, such as disuccinimidyl suberate, aldehydes, such as glutaraldehyde, bis-azido-compounds, such as bis / para-azido hexanediamine, bis-diazonium derivatives, such as bis- / para-diamonium-benzoyl / ethylenediamine, diisocyanates, such as tolylene 2,6-diisocyanate, and bis-active fluorine compounds, such as 1,5-difluoro- 2,4-dinitrobenzene.

 When used to inhibit the production of IgM antibodies by in vitro hybridomas, conjugates are generally added to the cell culture medium at a concentration of at least about 10 nM. The form and method of use in in vitro experiments do not play a decisive role. In general, aqueous forms are used that are compatible with the culture or perfusion medium. Cytotoxicity can be "read" using known techniques to determine the presence or concentration of IgM producing hybridoma cells.

 When used in vivo to inhibit the secretion of IgM cells, immunotoxins are administered to immunized animals in therapeutically effective amounts (i.e., amounts that exclude or reduce the production of IgM by splenocytes). They are usually administered parenterally or, preferably, by the intravenous route. The dose and dosage regimen will depend on the nature of the IgM producing cell to be suppressed, the characteristics of the particular immunotoxin, for example, its therapeutic value, and the activity of the action.

 The amount of immunotoxin used may vary from about 0.1 to about 10 mg / kg body weight.

 For parenteral administration, immunotoxins are used as a unit dose of injectable form (solution, suspension, emulsion) together with a pharmaceutically acceptable parenteral carrier. Examples of such carriers are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as fixed oils and ethyl oleate may also be used. As carriers, liposomes can be used. The carrier may contain small amounts of additives, such as materials that increase isotonicity and chemical stability, for example, buffers and preservatives. An immunotoxin is generally included in such carriers at concentrations from about 1 mg / ml to 1 mg / ml.

 Cytoxic radiopharmaceutical agents for suppressing IgM producing cells can be obtained by combining isotopes (e.g., V, Pt) emitting high linear energy carriers (PLEs) with antibodies. The term “cytotoxic moiety,” as used herein, includes such isotopes.

Labels that are used in the preparation of labeled antibody variants include components that can be detected directly, such as, for example, phosphors and radio tags, as well as enzymes that can form derivatives as a result of the reaction, which can then be detected . Examples of such labels are ³² P, ¹²⁵ I, ³ H, ¹⁴ C, fluorescent agents and their derivatives, rhodamine and its derivatives, dansil, umbelliferone, 2,3-dihydrophthalazine dione, ordinary horseradish peroxidase, alkaline phosphatase, lysozyme and glucose 6 -phosphate dehydrogenase. Antibodies can be labeled with such labels using known techniques. For example, coupling agents such as aldehydes, carbodiimides, dimaleimide, imidates, succinimides, bis-diazotized benzidine and the like can be used in order to provide antibodies with the above fluorescent, chemiluminescent and enzyme labels. Antibodies can also be labeled using magnetic beads used in the "magnetic selection" mode.

 Antibodies and labeled antibodies can be used in a wide variety of cell sorting procedures to separate IgM producing hybridoma cells from IgG producing hybridoma cells or to remove IgM producing hybridoma cells from cultures containing such cells.

 Techniques that can be used for analysis are known and include direct and indirect techniques. Direct assays include incubating a hybridoma or an unknown isotype antibody with a labeled antibody of the invention. If the sample includes IgM producing cells, the labeled antibody will bind to such cells. After washing the cells in order to remove unbound labeled antibodies, the sample is analyzed for the presence of labeled immune complexes. In indirect assays, a cell sample is incubated with an unlabeled monoclonal antibody. The sample is then treated with a labeled anti-monoclonal antibody antibody (e.g., labeled anti-rat antibody), washed and assayed for the presence of labeled tertiary complexes.

 The following examples provide a detailed description of the preparation, characterization and use of one of the representatives of the monoclonal antibodies of the invention. These examples should not be construed as limiting the present invention to any extent.

Example 1. The source and characteristics of anti-mouse IgM monoclonal rat antibodies
The rat hybrid, designated 58.6, was obtained from Dr. John Trial, Faculty of Immunology, Anderson Cancer Center. First, a 58.6 cell line secreted a rat antibody. The cell line was not stable and, after about four subcultures, 58.6 cells stopped producing any antibodies. The original source of cells was then cloned using limiting dilutions in order to obtain a cell line that was stable and continued to produce anti-IgM antibodies.

A) Cloning using limiting dilution
58.6 cells were cultured in Aiskouw environment for 3 days at a temperature of 37 ^o C in a humid atmosphere with 5% CO ₂ in air.

 When the propagating cell culture reached 50% fusion, they were collected by centrifugation and counted using a hemocytometer. Cells were diluted with 50% fresh medium and 50% treated medium (medium in which 58.6 cells were grown for 7 days) and placed on 96-well plates, about one cell per plate hole. When small colonies grew in the recesses containing one cell (approximately 12 days), the medium was removed and analyzed for anti-IgM antibody, as described in Example 2. Positive cells were expanded to significant volumes of antibodies, the cell material was frozen, and the antibodies were then subjected analysis and characterization. When the cell lines were fully analyzed for the type and specificity of the antibodies produced, the corresponding cell lines were again cloned and propagated to subsequently freeze the cell raw materials and inject Nistan treated nude mice to obtain ascites fluids.

B) Freezing hybrid cells
When the hybrid cells placed in T75 flasks reached 70% fusion, the cells were collected by centrifugation and the pellet was resuspended in 0.9 ml fetal calf serum. Immediately prior to freezing, the cells were transferred to freeze vials, and 0.1 ml of dimethyl sulfoxide (DMSO) was added to each vial. The vials were stored in liquid nitrogen.

C) Obtaining ascites fluids
Approximately 10 ⁷ cells of the hybridoma were washed in serum-free medium and injected intraperitoneally with a Nude mouse, which had previously received 0.5 ml pristan intraperitoneally 7-14 days before. Ascitic fluids generally formed within 1-3 weeks, and were collected from the abdominal cavity using a large calibration needle. The fluid was collected in tubes containing 5 ml of PBS (saline phosphate buffered) with 20 mM EDTA (ethylenediaminetetraacetic acid). After centrifugation at a speed of 2000 x g for 10 min, the supernatant was collected, treated with 0.1% sodium azide and stored at 4 ^° C or frozen in small portions at -20 ^° C. This fluid provided a rich source of monoclonal antibody (approximately 5-10 mg-ml).

Example 2. Selection and analysis of hybridoma cells producing rat anti-mouse antibodies
Hybridoma colonies, which were grown to a density of approximately 500-1000 cells for 2 weeks, were selected for subsequent analysis in order to determine which of the hybridoma cells produced antibodies that bind mouse IgM antibodies. The culture medium of these colonies was analyzed for the presence of rat anti-mouse IgM using an enzyme-linked immunosorbent assay carried out in accordance with the procedure of Boller et al. 100 mg 1 mg / ml purified mouse IgM or IgG (Sigma Chemical Company, St. Louis, Mo) was diluted in coating buffer (50 mM NaHCO ₃ , pH 9.8) and absorbed overnight on 96-well microtiter plates by incubation at 4 ^° C. in a humid chamber. Then the recesses were washed three times with phosphate-buffered saline containing 0.2% Tween-20 (PBS-Tween). After washing and removing all traces of liquid in the recesses by wetting with 100 μg supernatant, the hybridomas were added to the IgM-coated recesses and incubated at room temperature for 2 hours. The plates were washed again with PBS-Tween and incubated for 1 h at room temperature with 100 μl diluted in a ratio of 1: 1000 PBS-Tween goat conjugated with peroxidase anti-rat IgG. After this, the recesses were washed again, as described above, and then a reaction was performed with 100 μl of 1 mM ABTC (2,2-azino-di) 3-ethylbenzothiazoline sulfonic acid in 0.1 M citrate buffer, pH 4.2, containing 0 , 03% hydrogen peroxide for 20-60 minutes at a temperature of 37 ^o C. The optical density was measured in the range of 405 nm on a MikroElisa Reader.

 In order to assess whether the epitope recognized by anti-IgM antibodies was located on the antibody heavy or light chain, 9 hybridoma supernatants were bound to coated IgM-lambda and IgM-kappa plates. Hybridomas 1C2 and 2G10 bind equally well to the coated IgM-kappa and IgM-lambda depressions, which indicates that the binding specificity of anti-IgM antibodies is located on the heavy (mu) chain of the IgM antibody. The other 7 antibodies tested also bind to murine IgM, as indicated by the results in FIG. 1. As can be seen in FIG. 1 and 2, nine different hybridomas were tested against plates coated with either IgM kappa or IgM lambda. Standard ELISA assays were performed to evaluate rat immunization associated with each plate. It was found that all antibodies bind in depressions coated with both IgM kappa and IgM lambda. Among the antibodies with the highest binding, 1C2 and 2G10 were selected for further study. Given the best characteristics of the growth and production of antibodies, the 2G10 antibody was ultimately selected. It gave an optical density of 0.3 ± 0.03 (standard deviation). The reference point was 0.06 units of optical density (O.P.) when using medium without rat monoclonal antibody. Wells that gave a response to an anti-mouse IgM antibody greater than or equal to 0.3 O.P. were selected.

The specificity of the rat monoclonal 2G10 antibody was tested by ELISA under a wide variety of conditions. To determine the selective recognition of IgM compared to IgG using 2G10, 100 ng of mouse IgM lambda or IgG ₃ lambda was absorbed on microtiter plates (100 μl) and incubated with increasing concentrations of 2G10 antibody. As shown in FIG. 2, in the range of changes in antibody doses of 2-1000 ng, 2G10 was associated with IgM coated plates to a greater extent than with IgG coated plates. At 2G10 antibody doses of 50 ng or lower, no binding was observed on IgG-coated plates, while IgM-coated depressions were recognized by 2G10.

As shown in FIG. 3, the 2G10 antibody (added at a dose of 100 ng in 100 μl), incubated in recesses coated with IgG ₃ and IgM, indicates selective binding to coated IgM plates at all coating concentrations. Ten times more significant reactivity was measured by ELISA on IgM coated plates compared to IgG coated, at a coating dose of 10,000 ng IgM or IgG (compare IgG ₃ kappa to IgM kappa). This again confirmed selective IgM binding.

The 2G10 antibody was also tested for its ability to selectively recognize IgM in an indirect ELISA assay. Antigens were applied to microtiter plates and incubated with hybridoma culture medium that contained an antibody recognizing these antigens. Antibodies of subclasses IgM and IgG _{1 were} used in this assay. After incubation of the plates coated with the antigen with antibodies interacting with it, increasing concentrations of rat 2G10 antibody were added in the recesses, and 2G10 binding was determined by ELISA. As shown in FIG. 5, 2G10 sensibly and selectively recognizes IgM associated with its corresponding antigen (dark circles), but is not able to detect IgG (light circles) under the same conditions, although the presence of IgG ₁ could be easily detected by antibodies active against all subtypes of mouse immunoglobulin . These results confirm the selective recognition of mouse IgM subclass immunoglobulins using rat monoclonal antibody 2G10. The rat monoclonal antibody could also recognize the IgM antibody in picogram doses, which confirms its high affinity for the mouse IgM antibody.

Example 3. Characterization of rat anti-mouse IgM antibodies
Mouse immunoglobulin of various subtypes (IgM kappa and lambda, IgG ₁ , IgG ₂ and IgG ₃ ) were coated with microtiter plates, as in Example 2, and then the binding of rat anti-mouse IgM antibodies produced by hybridoma cells that were positive was studied in more detail in example 2. All plates were “read” in the region with a wavelength of 405 nm. Absorption (compared to control) was considered as an indication of the presence of antibodies against mouse IgM.

Example 4. Purification and characterization of rat monoclonal antibody 2G10
As shown in example 3, the antibody 2G10 gave a positive reaction with IgM-kappa and lambda. This antibody was purified by centrifugation and fractionation using ammonium sulfate.

 The hybridoma cell line designated 2G10 was deposited with the American Type Culture Collection (ATCC), Rockville, MD, USA on April 23, 1990, where it has storage code N HB 10436. These cultures are available in accordance with patent law and regulation. in this area in the United States of America and those foreign countries in which analogues of this patent application are filed. The accessibility of a deposited culture does not include permission to carry out the invention that is the subject of this patent application, as well as a patent granted to this patent application or any separate application, or its continuation.

The culture supernatant of 2G10 (or ascitic fluid obtained from Nude mice) was brought to 45% saturation with ammonium sulfate (salting out) by slowly adding an equal volume of a solution of ammonium sulfate with 90% saturation. The sample was stirred for 30 minutes at 4 ^° C and then centrifuged at a speed of 20,000 xg for 30 minutes. The precipitate was again suspended in a solution of ammonium sulfate at 40% saturation, stirred for 30 minutes and the precipitate was again obtained by centrifugation, as described above. This pellet was resuspended in water and dialyzed against 100 volumes of PBS. Portions of the solution were used to determine the protein content (by optical density at 280 nm), purity (using SDC-PAGE) and binding specificity (using ELISA). The remaining antibody solution was frozen at -20 ^o C and stored until use.

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

 The subclass of rat monoclonal antibody 2G10 was determined using the Ouchterloni procedure (Ouchterlony and Nilsson (1958) in Handbook of Exp. Immun., Ed. Weir, ed. Blackwell Scientific, London, p. 19.1-19.44) using an immunodiffusion reagent kit, manufactured by ICN Immunobiologicals (Lisle, IL).

The 2G10 antibody subclass is very important in determining the purification method. To perform the analysis of the subclass, a set of reagents for Ouchterloni immunodiffusion was used. In this case, antisera against various subtypes of rat immunoglobulin were placed in the corresponding recesses. A known standard or an unknown sample was added to the central recess and allowed to diffuse into a semi-solid medium. The deposition band at the site of a specific antiserum indicates a subtype. As shown in FIG. 7, positive control samples containing all rat subtype antibodies show reaction lines in all recesses (for all subtypes). On the other hand, the 2G10 antibody only interacted with the IgG _2A subtype antiserum, which means that the rat 2G10 antibody is an IgG _2a antibody.

 In order to evaluate the binding of commercially available rat antibodies to murine IgM, the activity of the rat monoclonal antibody L-O-MM-9 (Serotec, cat # MCA 199) against mouse IgM was evaluated in an ELISA test. One hundred nanograms of mu-kappa, gamma-kappa or gamma-lambda were placed in each of the recesses of the 96-recessed plate. Then, various amounts of rat antibody LO-MM-9 were added, and rat antibody ELISA was performed as previously described. As shown in the table. 1, no binding of this rat antibody to murine IgM-coated depressions was noted.

 Thus, this antibody does not appear to be useful for further study. In addition, as shown in FIG. 8-11 (lane 3), the separation of this antibody in SDS-PAGE gave at least three major protein bands and at least five weak protein bands.

Example 5. The binding of anti-mouse IgM rats with cells producing IgM
In order to confirm that rat 2G10 anti-mouse IgM antibody binds not only to a 96-well plate coated with purified IgM antibody, but also to IgM antibody producing cells, FACS analysis was performed on 10C1 cells and 238- hybridoma cells 57ADR mice that secrete IgG and IgM, respectively. For this, 1 • 10 ⁶ cells were centrifuged at a speed of 500 • g for 3 min, then washed three times with PBS, and then again suspended in 3 ml of PBS.

Purified on the basis of the affinity for fluorescein, the F (ab) ₂ fragment of the anti-mouse IgM goat IgM (Kappel company) was diluted in a ratio of 1: 100 in PBS (1x) and 20-40 μl of dilution was added to 20 μl of cell suspension. After incubation for 15-20 minutes in the dark at room temperature, the cells were washed twice with PBS, centrifuged at a speed of 500 rpm for 3 minutes. To fix the cells, a portion (300 μl) of paraformaldehyde (1% in PBS) was added. Cells were incubated at 4 ^° C. and then sorted by flow cytometry.

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

 As shown in table 2 and in FIG. 8-11, the 2G10 antibody is specifically bound to IgM contained on the surface of secreting IgM cells of mouse hybridomas, and is not related to secreting IgM cells.

 As shown in the table. 2, "background" fluorescein was not observed in untreated cells. Arbitrary rat IgG also did not bind to either IgG hybrids or IgM hybrids. No binding of the 2G10 antibody to IgG producing hybridoma cells was also noted (1.58% of the cells are positive, Fig. 8.9 and Table 2). However, as shown in FIG. 10 and 11 and in table. 2, 90% of the mouse IgM antibody producing cells bind strongly to the 2G10 antibody. Thus, the binding of the 2G10 antibody to the cells takes place due to the recognition of mouse IgM on the cell surface.

Cytotoxicity assay
The proposed antibodies were conjugated to the Ricin A toxin A chain (RTA) treated with LDPE, as described by Karlsson, J. et al. Biochem. Journ. (1978), vol. 177, pp. 723-727, or iminothiolan (IT).

Example 6. Connection 2G10 with gelonin
Received a raw solution of SPDP reagent (N-succinidyl-3- (2-pyridyldithio) propionate)) 6 mg / ml) in dry DMF. In a 1 ml PBS solution containing 1 mg of 2G10 antibody in a 12 x 75 mm glass tube, LDPE was slowly added up to a 5-fold molar excess (approximately 10 μl of the raw solution). The mixture was stirred every 5 minutes for 30 minutes at room temperature.

Excess unreacted LDPE was removed from the sample by gel filtration on a Sephadex G-25 column (Sephadex G-25) (1 x 24 cm), which was pre-equilibrated with 100 mM Na-phosphate buffer, pH 7.0, containing 0.5 mM EDTA (Buffer A). Fractions (0.5 ml) were collected and analyzed for protein content using Bradford dye binding assay (Bradford (1976) Anal. Biochem. T. 72, pp. 248-254). Absorbance (600 nm) was analyzed on a 96-well plate using a Bio-Tek Microplate type auto-reader. The antibody eluted in free volume (fractions 14-20), these fractions were collected and stored at 4 ^o C.

Gelonin toxin was extracted from the seeds of Gelonium multiflorum and subjected to purification to a uniform state using the procedure of Stirpe et al. (Stirpe et al. J. Biol. Chem., V. 255, pp. 6947-6953 (1980)). One milligram of purified gelonin (2 mg / ml in PBS) was added to HC1-triethanolamine buffer (TEA (HC1) to a final concentration of 60 mM TEA / HCl, and the pH was adjusted to 8.0. The solution was made containing 1 mM EDTA. Raw material was added. a solution of 2-iminothiolan - / 0.5 M in 0.5 M TEA / HCl, pH 8.0 / to a final concentration of 1 mm, and the sample was incubated for 90 min at 4 ^o C.

Excess reagent of 2-iminothiolan was removed by gel filtration on a Sephadex G-25 column (1 x 24 cm), which was previously equilibrated with 5 mM Tris-acetate buffer, pH 5.8, containing 50 mM NaCl and 1 mM EDTA. Fractions (0.5 ml) were collected and analyzed for protein content on 96-well microtiter plates using a Bradford dye binding assay. Gelonin was eluted in fractions 14-20; these fractions were collected and stored at 4 ^o C. Antibody 2G10 modified with SDP was mixed with a 5-fold molar excess of gelonin modified with 2-immunothiolane, the pH of the mixture was adjusted to 7.0 by adding 0.05 M TEA / HCl buffer (pH 8.0) and the mixture was incubated for 20 hours at a temperature of 4 ^o C in nitrogen atmosphere. Iodoacetamide (0.1 M in PBS) was added to a final concentration of 2 mM to block any remaining free sulfhydryl groups, and incubation was continued for another hour at a temperature of 25 ^o C.

Purification of 2G10 Gelonin Complexes
To remove low molecular weight products and unconjugated gelonin, the reaction mixture was applied to a Sephadex G-300 column (1.6 x 31 cm), pre-balanced with PBS. Fractions (1.0 ml) were collected and 50 μl portions were analyzed for protein content using a Bio-Rad dye binding assay. To remove unconjugated 2G10, the high molecular weight peak / fractions 17-23 / c of the G-300 column was applied to a Blue Sepharose GL-6B type affinity chromatography column, which was pre-equilibrated with 10 mM phosphate buffer (pH 7.2) containing 0, 1 M NaCl. After loading the sample, the column was washed with 30 ml of buffer until the unconjugated antibody was completely eluted. The column was eluted with a linear salt gradient of 0.1 to 2 M NaCl in 10 mM phosphate buffer, pH 7.2. The protein content in the eluted fractions was determined using the dye binding assay described previously.

 The “total” mixture containing 2G10 free antibody, 2G10-gelonin and free gelonin was first purified by gel filtration on a G-300 column. As shown in FIG. 12, a peak with a high molecular weight (fractions 25-42) was detected, as well as a peak with a lower molecular weight (fractions 55-67). Fractions 26-42 were collected for analysis on the purity and chemical activity of the conjugate.

 PAGE analysis of the purified 2G10 gelonin conjugate was performed. As can be seen in FIG. 13, the combined 2G10-gelonin mixture (lane 3) contained free 2G10, free gelonin (arrows), as well as 2G10 connected to 1 gelonin molecule (single arrow), and 2G10 connected to two gelonin molecules (double arrow). As you can sit on lane 1, the final purified 2G10-gelonin conjugate contains mainly 2G10 linked to 1 gelonin molecule and smaller amounts of 2G10 linked to 2 gelonin molecules. This drug was not contaminated with free gelonin or free antibody.

 To determine whether the chemical reaction and binding of the 2G10 antibody to gelonin was modified by recognition of the 2G10 antibody by mouse IgM, 96-well plates were coated with either a mouse IgG antibody or an IgM antibody, as in FIG. 3. Instead of 2G10 antibody, 2G10-gelonin conjugate was added to the wells in various concentrations. To detect a rat antibody, a standard ELISA assay was then performed. As shown in FIG. 14, the 2G10-gelonin conjugate readily binds to IgM and only to a small extent with IgG coated plates. Only at the highest test concentrations (1000 ng) did the 2G10-gelonin indentation / conjugate bind significantly to IgG-coated depressions. In contrast, at a similar concentration, the 2G10-gelonin conjugate was substantially bound to IgM-coated depressions. Thus, the immune activity of the 2G10-gelonin conjugate against IgM is maintained. In addition, the 2G10-gelonin conjugate did not cross-react to any noticeable extent with murine IgG, and thus, the selectivity of the 2G10-gelonin conjugate also did not change. These data indicate that the conjugate 2G10-gelonin should bind to IgM covering mouse cells, according to the same scheme as the 2G10 antibody itself.

Example 7. The production of ODS-monoclonal antibodies
A. In vitro Immunization and Production of Monoclonal Antibody
The descriptions currently used in the preparation of murine monoclonal antibodies provide for the formation of hybridomas that secrete antibodies of the IgM subclass. In order to confirm the most general result obtained in accordance with these descriptions, two methods were used to form monoclonal antibodies against rat ornithine decarboxylase (ODS). The first technique involves immunization of mouse spleen cells in vitro, i.e., in culture dishes, with ODS protein.

 This procedure was carried out as described by Luben et al. (Luben and Mohler (1980), Molec. Immunol, v. 17, pp. 635-639) using 25 mg of purified rat liver od.

ODS protein was incubated with mouse cells for 72 hours at 37 ^° C in the presence of thymocyte-conditioned medium (a source of special growth factors for cells secreting immunoglobulin). The spleen cells were then fused to MRS myeloma cells using polyethylene glycol as a fusion stimulating agent. The resulting hybrid cells were tested for the secretion of an antibody chemically active with respect to the ODS protein. The results are shown in table. 3.

 After the initial screening with ODS protein, the hybridomas were again cloned and tested again. Seventy hybridomas were obtained which interacted with the ODS protein according to several criteria. However, as the table shows. 3, all clones secreted IgM isotype antibodies. None of them secreted IgG antibodies. These antibodies have been found to have limited use in a wide variety of monoclonal antibody applications.

 B. In vivo immunization and production of monoclonal antibodies.

 The second technique used to produce monoclonal antibodies was to immunize mice by injecting purified ODS protein. Murine spleen cells were isolated after immunization, fused with P3 • 63 Ag 8.653 myeloma cells (using PEG, as described above). Clones were isolated, tested for chemical activity with respect to the ODS protein, and then characterized for use as a specific ODS - recognition reagent. The results of these tests are given in table. 4.

 Twenty-seven monoclonal cell lines were propagated, 100% of which secreted an IgM subclass antibody. It has been repeatedly shown that these antibodies are inadequate for use as ODS-specific reagents.

 Example 8. Flow activated cell sorting (FACS procedure).

 IgM secreting cells of hybridoma 238-57ADR were centrifuged at a speed of 500 xg for 3 min, washed three times with PBS and resuspended in 3 ml of PBS.

The fluorescein-conjugated, affinity-purified F (ab) ₂ IgM anti-mouse immunoglobulin fragment (Kappel) was diluted in a ratio of 1: 100 PBS, and 20-40 μl was added to 20 μl of cell suspension.

 After incubation for 15-20 minutes in the dark at room temperature, the cells were washed twice with PBS, centrifuged at a speed of 500 rpm for 3 minutes.

A 300 μl portion of paraformaldehyde (1% in PBS) was added to fix the cells. Cells were incubated at a temperature of 40 ^o C before sorting using flow cytometry.

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

 Example 9. Magnetic separation of cells.

Mouse 238-57 ADR hybridoma cells were washed three times in Iskov medium containing 1% fetal calf serum and 0.1% gentamicin. Cells were separated in a centrifugal separator at 500 x g for 3 min at room temperature and then counted. The pellet was resuspended in medium (0.5 ml) and incubated for 60 min on ice with purified rat anti-mouse IgM antibody (2G10) at a concentration of approximately 0.425 mg / ml (about 0.1 ml was used per 10 ⁶ cells / ml). After washing twice in cold Iskova medium, the cells were resuspended in 0.2 ml of medium and counted. Magnetic granules were washed three times in serum free medium using a magnetic plate. The cell pellet was mixed with pellet pellet at a ratio of 20 pellets / cell. The total volume should not exceed 0.4 ml. The cell / pellet mixture was incubated 1/2 hour on ice with stirring every 10 minutes.

 The cell / granule mixture was resuspended in at least 2 ml of medium and separated magnetically in a direction perpendicular to gravity. After completion of the separation, the supernatant was removed without affecting the magnetic deposit. The granules were resuspended in 1-2 ml of medium and examined microscopically.

Example 10. The study of lymphocytes conjugate 2G10-gelonin
To demonstrate the cytotoxicity of the 2G10 gelonin conjugate, two female Balb (c) mice (Harlan Sprague Dawley) were used, in which the spleens were excised. In a laminar flow box, the skin over the spleen was washed with 70% isopropyl alcohol and opened with aseptic or sterilized scissors. Using a pair of sterile tweezers and scissors, the peritoneum was opened and the spleen was exposed. The spleen was placed in a 100 ml sterile Petri dish with 10-15 ml of culture medium (Iscoves; Gibco) with 10% fetal calf serum (Hyclone) and 50 g / ml of gentamicin (Tri-Bio Laboratories). The spleen was washed in the medium and then transferred to a clean Petri dish with fresh nutrient medium and closed.

Lymphocyte preparation
In a laminar flow box using sterile instruments, excess fat and connective tissue (white material) were cut off from the spleen. Spleen tweezers were transferred to a new Petri dish with 20 ml of culture medium. The washing procedure was repeated twice. The spleens were dispersed using two 10 ml syringes with calibrated needles until large fragments of tissue remained. The spleen cells (splenocytes) had the appearance of opaque mixtures in a nutrient medium and contained both red and white cells. Cells and medium were pipetted into a 50 ml centrifuge tube (Corning), and the remaining large undispersed tissue pieces were precipitated. Cells were placed on ice in a closed tube for 10 min.

The upper portion was selected with a pipette (leaving larger sections of undispersed tissue), and centrifuged for 10 min at a speed of 1800 revolutions per minute (rpm) in a centrifuge with a Dupon Sorvall 2LC-2B upper platform; the supernatant was drained. The pellet was resuspended in 10 ml of culture medium and the cells were counted using 5 μl of cell suspension and 95 μg of trypan blue solution in 1: 2 dilution using a hemocytometer and a Nicon phase contrast microscope with a 100-fold increase. The total cell miscalculation was 6.24 • 10 ⁸ cells. The solution was adjusted to 20 ml to obtain 3.12 • 10 ⁷ cells / ml of culture medium. One ml of this cell suspension was added to several 15 ml centrifuge tubes.

 Sample group 1: only control medium was added to the cells; group 2: only control medium was added to the cells; group 3: only control medium was added to the cells; group 4: 2G10 antibody (AB) with a concentration of 1 mg / ml; group 5: 2G10 antibody with a concentration of 0.1 μg / ml; group 6: 2G10 antibody with a concentration of 0.01 μg / ml; group 7: gelonin with a concentration of 1 μg / ml, equivalent = 0.2 mg / ml; group 8: gelonin with a concentration of 0.1 μg / ml, equivalent = 0.002 μg / ml; group 9: gelonin with a concentration of 0.01 μg / ml, equivalent = 0.02 μg / ml; group 10: Ab + gelonin with 5 μg / ml Ab + 0.1 g / ml gelonin; group 11: Ab + gelonin with 0.05 μg / ml Ab + 0.02 μg / ml gelonin; group 12: Ab + gelonin with 0.005 μg / ml Ab + 0.002 g / ml gelonin; group 13: conjugate 2G10 + gelonin with 1 g / ml; group 14: conjugate 2G10 + gelonin with a concentration of 0.1 μg / ml; and group 15: conjugate 2G10 + gelonin with a concentration of 0.01 μl / ml.

Protein was added to the appropriately numbered tubes and the total volume in each was adjusted to 5 ml with Iskov culture medium. The lids were ajar and the tube was incubated for 24 hours at 37 ^° C in a CO ₂ incubator (Queue) (5% CO ₂ - 95% air). Then, centrifugation was performed at a speed of 1500 rpm for 10 min in an IEC Centra-7R centrifuge at 4 ^° C. The supernatant was removed and stored. The cell pellet was resuspended in 10 ml of culture medium and centrifuged. The supernatant was poured, and the cells were washed twice with 4-5 ml of culture medium by centrifugation. The treated cells were transferred into 5 ml of culture medium on a 6-well plate (Falcon) and incubated at 37 ^° C. in a CO ₂ incubator. Subsequently, on certain days, 300 μl of culture medium was collected in a numbered microcentrifuge tube. 1 μl of 10% sodium azide was added to each such tube and the tubes were left at 4 ^° C. Samples of the culture medium were taken after 24 hours, 72 hours, 96 hours. A solid phase immunosorbent assay was used to determine the effect of the treatment on the secretion of IgM from the primary lymphocyte culture .

50 μl of 2 mg / ml goat anti-mouse IgG (solution 2 mg / ml) (Sigma) was placed in 12 ml of double-distilled water (dd H ₂ O). Using a multichannel pipette (Titer-Tek), 50 μg of this solution was placed in each well of a 96-well microtiter plate (Falkon), with 500 μl of goat anti-mouse immunoglobulin G antibody placed in each well; then, drying was performed by incubation overnight in an open form in an oven at 37 ^° C. 100 μl of 5% bovine serum albumin (BSA) in physiological saline with phosphate buffer (PBS) was added to each well of a 96-well plate and incubated 1, 5 hours at room temperature. Liquid was removed by tipping the tablet onto paper towels. Each well of a 96-well plate was washed with PBS (1x) containing Tween-20 (0.05%) (Biorad), and the liquid was removed by tipping the plate on dry paper towels.

100 μl of each supernatant was added to individual wells of a 96-well plate and incubated for 1.5 hours at room temperature. The wells were washed three times with PBS / Tween-20, 100 μl goat antibody was added against the mouse immunoglobulin mu chain chemically conjugated to horseradish peroxidase (Sigma) in a 1: 1000 dilution (in 1 mg / ml BSA in PBS), and incubated 1.5 h at room temperature. Added 100 μl ABTS / / 2,2 ^1- asino-bis / 3-ethylbenzothiazoline-6-sulfonic acid // (Sigma) in 0.1 M citrate buffer (pH 4.2) with 0.03% hydrogen peroxide ( 11 μg of 30% H ₂ O ₂ in 11 ml of ABTS), and after 20 minutes of incubation at room temperature, the plate was placed in a Bio-Tek Laboratories autocounter to read the absorbance value. The conjugate of the present invention significantly inhibited the secretion of IgM from primary lymphocytes after 24 hours (FIG. 11), 48 hours (FIG. 16) and 72 hours (FIG. 17). The results shown in FIG. 15-17 illustrate that dose-dependent conjugate treatment reduced IgM levels in these primary lymphocyte cultures. Other treatments failed to alter IgM levels in any of the dosages that were tested. Therefore, the level of IgM produced by lymphocytes was suppressed by the covalent association of anti-mouse IgM monoclonal antibody 2G10 with gelonin.

Example 11. The study of lymphocytes # 2 conjugate 2G10-gelonin
The procedure was the same as in Example 10, except that the final protein concentrations were 5 μg, 0.5 μg and 0.05 μg, or the equivalent molar concentration for gelonin.

 In FIG. 18-20 show that the conjugate of the present invention strongly inhibited the secretion of IgM from primary lymphocytes after 24, 48 and 72 hours, respectively.

Example 12. The study of lymphocytes # 3 conjugate 2G10-gelonin
Cells were prepared as in examples 10 and 11, except that they were open for processing (control, antibody, gelonin, Ab + gelonin and conjugate) by incubation for 2 hours on ice. Then the cells were centrifuged and washed twice with medium, transferred to 6-well plates and incubated. Samples of the supernatant were collected at 24, 48, 72 and 96 hours intervals. The final protein concentration was 5 μg / ml (or equivalent for gelonin). A new 2G10-gelonin conjugate was obtained, and it was tested with the first conjugate.

 In FIG. 21-24 show that the conjugates of the present invention clearly inhibit the secretion of IgM antibodies from lymphocytes after 24, 48, 72 and 96 hours, respectively. Obviously, a short incubation with the immunoconjugate is enough to obtain changes in IgM secretion from cells.

Example 13. The fusion of murine spleen cells (Balb / c) with murine myeloma cells (P3 x AG63 x 8)
The nutrient medium for P3 x AG63 x 8 myeloma cells was Iskova medium (Gibco) with 500 ml containing 10% fetal calf serum (Hyclone) and 0.5% ml of gentamicin (Trio-Bio Laboratories).

A solution of 8-azo-guanine (20 μl / ml) was replaced with the usual nutrient medium for (P3 x AG63 x 8) myeloma about a week before the time of fusion. The HAT solution consisted of 0.5 ml of gentamicin, 5 ml of 100 • HT, 5 ml of 100 • A and 20% B (fetal calf serum). A solution of 100 • HT consisted of 136 mg of hypoxanthine (10 mM) and 39 mg of thymidine (1.6 mM) in 100 ml of dd H ₂ O (heated to 70-80 ^o C). 100 • A consisted of 1.8 mg of aminopterin (100 ml of H ₂ O, pH 7.8).

 The fusion was carried out after preliminary washing of the spleen cells in serum and gentamicin-free medium by centrifugation for 5 min at 200 • g (1000 rpm in a centrifuge with an upper Sorvall CLC-2B platform). After resuspension in 10 ml of serum-free medium, the cells were diluted 1:20 (5 μl in 100 μl of medium) and counted in a hemocytometer.

Used flasks with a merging layer, 50-60% filled with P ₃ myeloma cells; cells were collected by centrifugation and washed 4 times in serum free medium (5 min at 500 x g), and miscalculated. Spleen and P ₃ cells were combined in a 4: 1 ratio (8 • 10 ⁷ spleen cells and 2 • 10 ⁷ myeloma cells) and pelleted together by centrifugation for 5 min at 1200 rpm. 1 ml of a cold solution of polyethylene glycol-4000-dimethyl sulfoxide / PEG-400-DMSO / was added, and the cells were incubated in this mixture for 15 s, then slowly diluted with PHC-DMSO, adding 20 ml of serum-free medium dropwise over a 3-minute period. . The cell mixture was centrifuged at 400 x g (166 rpm) for 6 minutes to separate the PEG diluent. Cells were resuspended in 50 ml of 1 • HAT and pipetted into five 96-well plates (Falcon) at 100 μl / well and incubated. After plating the cells, they remained in the incubator for 3-4 days until 100 μl of fresh HAT medium was added, then they remained until the colonies formed.

Example 14. Testing IgM and IgG
When the supernatants of the fusion cells turned yellow, the secretion of IgM and IgG was determined using an ELISA assay. In this case, rigid 96-well microtiter plates (Falcon) were coated (500 ng / well) with a goat anti-mouse IgG antibody using a multichannel pipette and dried overnight in open form at 37 ^° C. The solution was blocked with 5% BSA in PBS (addition 100 μl to each well and incubation for 1.5 hours at room temperature). After washing with 3 PBS containing 0.05% Tween-20 (Bio-Rad), 50 μl was pipetted into each well on two separate plates (one for IgG testing and the other for IgM testing). A replacement culture medium was added to the fusion plate, and it was incubated for 1.5 hours at room temperature until washing with 3x PBS-Tween-20. The secondary antibody tested for IgM was a goat x mouse IgM antibody (specific peroxidase (Sigma)) diluted 1: 1000 in 1 mg / ml BSA in 1 x PBS. A secondary antibody was added to the respective plates and incubated for 1.5 hours at room temperature, and then washed 3 times with PBS-Tween-20. Detection was carried out by adding 100 # ABTS / 2,2 ^1- azino-bis / 3-ethylbenzothiazolin-6-sulfonic acid / containing 0.03% hydrogen peroxide; the plates were read on a Bio-Tek Instruments 405 nm auto-plate reader.

Example 15. The fusion of murine myeloma cells (P3 x AG63 x 8) with spleen cells of treated female Balb / c mice
Groups are processed as follows. Group 1: control mice - PBS; group 2: antibody 2G10 and gelonin; and group 3: conjugate. The conjugate was administered intraperitoneally at a concentration of 500 μg 500 μl PBS on the 1st and 4th days. A mixture of 2G10 antibody and gelonin was administered to mice at a concentration of 250 μg of antibody + 50 g of halonin in 500 μl of PBS on days 1 and 4. The control injection was done intraperitoneally in a volume of 500 μl on the 1st and 4th days. On the 9th day, the spleens were dissected and the cells were prepared as described above. Then, each spleen sample was fused with an equal number of myeloma cells using 8 • 10 ⁷ spleen cells and 2 • 10 ⁷ myeloma cells for each fusion. The supernatants were tested a week later for the secretion of IgG and IgM antibodies in each group and evaluated. The percentage of viable colonies secreting IgM and IgG and those wells that contained both IgM and IgG antibodies are shown in FIG. 21. Obviously, IgM is the main form of immunoglobulin secreted from viable cells obtained from animals that were injected with saline or a mixture of 2G10 and gelonin. Immunoconjugate treatment significantly increased (from 3 to 30%) the number of IgG secreting colonies and IgG and IgM secreting colonies (this is confirmed by the specific detection of IgG and IgM in the supernatant wells of the used culture containing colonies). Thus, the immunoconjugate increases the number of viable colonies and the secretion of IgG by viable colonies after cell fusion. These results show the effect of an in vivo immunoconjugate on the expression of immunoglobulin in a healthy animal after immortalization of their lymphocytes by cell fusion. Immunoconjugate treatment of animals would be useful in inducing in vitro production of IgG following conventional cell fusion immortalization processes in monoclonal antibody production schemes.

Example 16. Detection of IgM in the serum of pre-treated KLH-immunized mice 1
Female Balb / c mice were treated and immunized as follows. On day 0, 150 μl of blood was taken from the tail vein of each mouse into a 0.5 microcentrifuge tube, centrifuged for 10 minutes, and the distilled serum was carefully pipetted into a clean centrifuge tube marked. These tubes were stored at -20 ^° C until analysis. After blood sampling, the first treatment was done. Mice were divided into 4 groups (2 mice per group). The first group was a control and received 500 μl of saline with phosphate buffer intraperitoneally; the second group also received an intraperitoneal injection of 500 μg of 2G10 antibody in PBS (500 μl). The third group received 100 μg gelonin (molar equivalent dose of 500 μl) in 500 μl PBS and the fourth group received an intraperitoneal injection of 2G10-gelonin conjugate (a dose of 500 μg in 500 μl PBS was administered intraperitoneally).

 On the second day, each mouse was subjected to a second treatment, which repeated the first treatment, but with a half dose. All injections were done intraperitoneally. On day 4, the third treatment was the same as the second. On the 5th day, blood was taken from each mouse for analysis and the first immunization was done [the chromoprotein obtained from the mollusk Megathure Crenulata, KLH (Keyhole-limpet-hemocianin) / DNP (dinitrophenol)]. A dose of 250 μg DNP-KLH in 200 μl PBS was injected intraperitoneally into each mouse. On the 6th day, the fourth treatment was done intraperitoneally: to control mice - 125 μl PBS; antibody 125 μl in 125 μl PBS; gelonin - 50 μl in 125 μl PBS; conjugate - 125 μl in 125 μl PBS.

 On the 8th day, the fifth treatment was done. The dose was the same as in the fourth treatment. On the 10th day, blood was taken for analysis. Then the sixth treatment was carried out, which was the same as the fifth. On day 12, a seventh treatment was performed. The dose was 250 μg 250 μl, and control mice received 250 μl PBS. On day 13, a KLH booster was introduced. The booster was the same as in the first immunization. On the 20th day, blood tests were taken.

On day 20, serum samples were analyzed. To test for serum IgM in pre-treated - immunized KLH mice, four 96-well plates were coated with 150 ng DNP-KLH per well in a volume of 50 μl. Using a multichannel pipette, 50 μl was added to each well in four plates; they were left open and dried overnight at 37 ^° C. The plates were blocked with 100 μl / well of 5% BSA in PBS and incubated for 1.5 hours at room temperature. After washing (3 x PBS Tween-20), each serum sample was diluted 1: 500 (2 μl / 1 ml). 1 mg / ml BSA in PBS. 100 μl was injected into each well and incubated for 1.5 hours at room temperature. After washing (3 x PBS Tween-20), 100 μl of a secondary antibody, goat • mouse IgM peroxidase (Sigma), was added. The sample was diluted 1: 2000 with 1 mg / ml BSA-PBS and incubated for 1.5 hours at room temperature. After washing (3 x PBS Tween-20), 100 μl ABTS with 0.03% H ₂ O ₂ was added and read at 405 nm in an auto microplate reader. In FIG. 26 shows that the conjugate of the present invention suppresses the IgM response to KLH in vivo, demonstrating the effectiveness of the immunoconjugate in animals.

Example 17. Detection of IgM in the serum of pre-treated KLH-immunized mice II
Female Balb / c mice were treated and immunized as follows. On day 0, each mouse was taken and left blood for analysis. On day 1, the mice were divided into 4 groups: group 1 was a group of control mice that received 500 μl of saline in phosphate buffer intraperitoneally; group 2 received 100 μg (equivalent dose of 500 μl) of gelonin in 500 μl of PBS by intraperitoneal injection; group 3 received an antibody-gelonin mixture consisting of 250 μg of a 2G10 antibody and 50 μg of gelonin in 250 μl and PBS intraperitoneally; group 4 received 500 μg of conjugate 2G10 - gelonin in 500 μl of PBS also intraperitoneally. On the 4th day, a second treatment was done. On day 5, KLH immunization was performed, consisting of 250 μg KLH-DNP in 200 μl PBS. On the 12th and 18th days, blood was taken. On the 19th day, the third treatment was done (the same as the first and second). On the 20th day, a KLH booster was introduced (the same as during the first immunization). On the 29th day, blood was taken. On day 36, serum was tested for IgM in pre-treated KLH immunized mice. In FIG. 27 shows results that are similar to those of FIG. 26.

 The presented examples, together with the methods, procedures, designations and specific compounds described herein, are intended to illustrate preferred options and should not be construed as limiting the scope of the invention. Those skilled in the art will appreciate that changes and other uses are possible without departing from the scope of the invention as defined by the claims.

Claims (8)

 1. The conjugate based on an anti-IgM antibody, comprising the cytotoxic agent gelonin associated with a monoclonal antibody that selectively interacts with an IgM antibody or with hybridoma cells producing an IgM antibody, does not interact with an IgG1 and IgG2 antibody and refers to G -isotype.  2. The conjugate according to claim 1, characterized in that the monoclonal antibody is obtained by culturing a hybridoma 2G10 or 1G2.  3. The conjugate according to claim 1, characterized in that the monoclonal antibody further comprises a label for its detection.  4. A method of reducing the secretion of IgM antibodies from lymphocytes, which consists in contacting the lymphocytes with a cytocidally effective amount of the conjugate described in claim 1.  5. An anti-JgM antibody conjugate comprising a gelonin cytotoxic agent coupled to a monoclonal antibody that selectively interacts with mouse IgM antibody or hybridoma cells producing antibody to mouse JgM antibody, does not interact with IgG1 and IgG2 antibody and relates to the G isotype.  6. The conjugate according to claim 5, characterized in that the monoclonal antibody further comprises a label for its detection.  7. The conjugate according to claim 5, characterized in that the monoclonal antibody is obtained by culturing a hybridoma 2G10 or 1G2.  8. A method of reducing the secretion of mouse IgM antibodies from lymphocytes, which consists in contacting the lymphocytes with a cytocidally effective amount, as described in paragraph 5.

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