EP0535084A1

EP0535084A1 - Improved ascites production of monoclonal antibodies using an immunosuppressive anti-cd4-related antibody

Info

Publication number: EP0535084A1
Application number: EP19910911687
Authority: EP
Inventors: Timothy James Gorzynski
Original assignee: EI Du Pont de Nemours and Co
Current assignee: EIDP Inc
Priority date: 1990-06-22
Filing date: 1991-06-07
Publication date: 1993-04-07
Also published as: JPH05507851A; CA2085586A1; WO1992000100A1

Abstract

Procédé d'amélioration de la production ascitique d'anticorps monoclonaux consistant à immuno-déprimer des animaux, chez lesquels on fait se multiplier des cellules d'hybridome histoincompatibles, en leur administrant une quantité, qui permette d'obtenir une immunodépression, d'au moins un anticorps immunodépresseur apparenté aux anticorps anti-CD4.Method for improving the ascites production of monoclonal antibodies consisting in immunosuppressing animals, in which histoincompatible hybridoma cells are multiplied, by administering an amount thereof which makes it possible to obtain immunosuppression, at least minus an immunosuppressive antibody related to anti-CD4 antibodies.

Description

Improved Ascites Production of Monoclonal .Antibodies Using An Immunosuppressive Anti-CD4-Related Antibody

FIELP OF THE INVENTION This invention relates to the production of monoclonal antibodies in vivo by growing hydridoma cells as an ascites tumor and, more particularly, to improved ascites production of monoclonal antibodies using at least one anti-CD4-related immunosuppressive antibody.

BACKGROUND OF THE INVENTION Fifteen years have passed since George Kohler and Cesar Milstein first described somatic cell hybridization to generate a continuous "hybridoma" cell line producing a monoclonal antibody. (Nature 256: 495-497 (1975)). This technology fostered the production of thousands of different monoclonal antibodies which are useful for a plethora of scientific and commercial applications as described in Monoclonal Hybridoma Antibodies: Techniques and Applications, Hurrell (ed.), (1982) or Monoclonal Antibody Production Techniques and Applications, Schook (ed.), Immunology Series Vol. 33 (1987) and references cited therein.

Hybridomas can be grown in vivo as an ascites tumor in animals having the same genetic composition as the hybridoma cells, i.e, syngeneic. The serous (ascites) fluid which accumulates in the peritoneal cavities of these animals is collected using any of a number of conventional techniques.- See, e.g., Brodeur et al., J. Immunological Methods 71: 265-272 (1984) and Chandler, Commercial Production of Monoclonal Antibodies: A Guide for Scale Up, Seaver (ed.), pages 75-92 (1987) . This procedure works reasonably well provided the donor of the immune lymphocytes and the myeloma cells used to create the hybridoma cells are syngeneic with the animal in which the hybridoma cells will be grown. However, if there are antigenic differences between the source of the hybridoma cells and the host in which they will be grown, then no ascites tumor will develop due to the im unological intolerance of the animal, i.e., histoincompatibility, for the hybridoma cells. Thus, modifications of the standard procedures for ascites fluid production are needed when histoincompatibility exists.

Histoincompatible cells can be grown as an ascites tumor by first injecting the animals to be used for ascites production with hydrocortisone acetate or hydrocortisone succinate and then irradiating the animals with sublethal total body gamma radiation before injecting the hybridoma cells as was described by Weissman et al., J. Immunology, 135: 1001-1003 (1985). This procedure requires access to a source of gamma radiation which, if available, is quite time consuming to administer. In addition, animals exposed to this radiation are more prone to illness than animals which have not been exposed. Also, these animals have substantially shortened life spans.

Witt et al., Allerg. Immunol., 33(4): 259-264 (1987), describe the use of irradiation to elicit the production of ascites fluid of histoincompatible hybridoma cells which express cell surface neoantigens and/or tumor associated antigens or altered antigen patterns. This procedure suffers from the same limitations as those described above for Weissman et al. Another approach has been to mate the strains of mice donating immune lymphocytes with the strains of mice donating myeloma cells so that the offspring (Fl hybrids) have the same genetic composition as the hybridoma cells being injected thereby preventing rejection of the hybridoma cells. Gorzynski et al., J. Immunogenetics 12: 267-279 (1986). Unfortunately, this approach is limited. Fl hybrid mice may not be readily available and, thus, may have to be bred. Furthermore, if the donors of the immune lymphocytes and myeloma cells are of different species, then viable offspring will not be created because the technology may not be available to breed such species.

Abrams et al., J. Immunol. 132:1611 (1984), describe growing histoincompatible hybridomas in immunodeficient athymic mice. Such mice are not always commercially available. When they are available, they can cost four to five times more than thymus-containing mice. Because athymic mice are more susceptible to disease than thymus-containing mice, they require much more attention.

In vitro culture techniques have been used to produce large amounts of monoclonal antibody. One approach has been to grow the hybridoma cells in a fermentor as described by Reuveny et al., J. Immunological Methods 86: 61-69 (1986) . Using this approach, antibody concentrations in the range of 0.390 mg/mL of culture can be obtained. However, this is far removed from the 1.8-17.3 mg/mL antibody concentrations which are obtained using the ascites approach.

A number of monoclonal antibodies have been described as having immunosuppressive potential including, but not limited to antibodies which are capable of binding to the cell surface molecule, CD4 , which is a T-lymphocyte molecule referred to as L3T4 in mice. See, for example, Wofsy et al., Immunol. Res. 5: 97-105 (1986) . U.S. Patent 4,681,760, issued to Fathman on July 21, 1987, describes a method to suppress undesired immune responses such as allergic reactions, by co-administering the antigen for which immuno-tolerance is sought and an antibody which is specific for L3T4- equivalent differentiation antigen on T cells, thus, crippling the helper T cells which participate in the immune response.

Shizuru et al., Science, pages 278-280, Vol. 237 (July 17, 1987) describes islet allograft survival in diabetic mice which at the time of engraftment the mice received a single course of treatment with a monoclonal antibody directed against the L3T4 determinant. Jacob et al., J. Exp. Med. 166: 798-803 (1987) describe antibodies to cellular products such as gamma interferon as having immunosuppressive potential.

Similarly, Soulillou et al.. Lancet 1(8546): 1339-1342 (1987) , describe antibodies to interleukin-2 as having immunosuppressive potential.

The immunosuppressive potential of these antibodies has been identified under such conditions as autoimmunity (Kantwerk et al., Clin. Exp. Immunol. 70(3): 585-592 (1987)), allograft rejection (Mottram et al.. Transplantation Proceedings 19(1): 582-585

(1987)), resistance to metastatic tumors (Schild, Eur. J. Immunol. 17: 1863-1866 (1987)), and tolerance induction (Gutstein et al., J. Immunol. 137: 1127-1132 (1986) and Goronzy et al., J. Exp. Med. 164: 911-925 (1986)).

Monoclonal antibodies that define T-lymphocyte subsets in the rat are discussed in Monoclonal .Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennett et al. (eds.), pages 251-273 (1980). It was observed that one of the subset specific antigens may play an important role in immune functions, since the monoclonal antibody against it inhibited mixed lymphocyte reactions.

U.S. Patent 4,381,292, issued to Bieber et al. on April 26, 1983, describes monoclonal antibodies specific for an antigen diagnostic for thymocytes, normal peripheral T cells and some null cells. These antibodies distinguish among subpopulations of T cells and can be used in assays, cell sorting and immunosuppression. For example, they can be used as i munosuppressants for allograft recipients, either of the same or different species from mammalian lymphocytes used to prepare the hybridomas.

Roitt et al.. Immunology, pages 24.8-24.9 (1985) , describe immunosuppressive measures adopted to prevent chronic rejection reactions of an allogeneic transplant. One of these measures is antigen specific and involves the induction of anti-idiotypic antibodies to the T cells which recognize the graft. These antibodies block the recognition of the MHC antigens on the graft.

U.S. Patent 4,624,925, issued to Kung et al. on November 25, 1986, describes a hybrid cell line for producing monoclonal antibody to an antigen found on approximately 10% of normal human thymocytes. It is mentioned in column 6 at lines 24-68 that once the desired hybridoma has been selected and cloned, the resultant antibody can be produced in one or two ways: (1) the purest monoclonal antibody is produced by in vitro culturing of the desired hybridoma in a suitable medium for a suitable length of time, or (2) the desired hybridoma may be injected into mice, preferably syngeneic or semi-syngeneic.

U.S. Patent 4,621,050, issued to Sugimoto on November 4, 1986, describes a process for the production of human colony-stimulating factor. It is mentioned in column 3, line 57-through column 5, line 37 that the animals in which the hybridoma cells are grown can be treated, prior to cell transplantation, with irradiation, of about 200 to 600 rem of X-ray or gamma- ray, or with injection of antiserum or immunosuppressive agent prepared according to conventional methods.

U.S. Patent 4,537,852, issued to Sugimoto on August 27, 1985, describes a process for the production of human urokinase which is s.imilar to that described above for the production of human colony-stimulating factor.

SUMMARY OF THE INVENTION This invention concerns a process for improving ascites production of monoclonal antibodies which comprises i munosuppressing a host in which histoincompatible hybridoma cells are grown by administering an immunosuppressive amount of at least one immunosuppressive anti-CD4-related antibody.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph comparing the total amount of ascites fluid obtained from BALB/c mice treated with an immunosuppressive anti-CD4-related antibody versus the amount of ascites fluid obtained from untreated BALB/c mice in which histoincompatible hybridoma cells were grown.

Figure 2 is a graph comparing ascites production in B.ALB/C mice treated with an immunosuppressive anti-CD4-related antibody versus the amount of ascites fluid obtained from untreated BALB/c mice in which histoincompatible hybridoma cells were grown. Figure 3 is a graph comparing ascites production in Fl mice treated with an immunosuppressive anti-CD4-related antibody with ascites production in untreated Fl mice in which histoincompatible hybridoma cells were grown.

Figure 4 is a graph comparing ascites production in Fl mice treated with an immunosuppressive anti-CD4-related antibody with ascites production in untreated mice in which histoincompatible hydridoma cells were grown.

Figure 5 is a graph depicting the poor results obtained when non-anti-CD4-related monoclonal antibodies having immunosuppressive potential are used.

Figure 6 is a graph comparing ascites production in BALB/c mice treated with an immunosuppressive anti-CD4-related antibody and/or cyclophosphamide with ascites production in untreated mice in which histoincompatible hybridoma cells were grown. Figure 7 is a graph comparing ascites production of a histoincompatible hybridoma cell in BALB/c mice treated with an immunosuppressive anti-CD4- related antibody with ascites production in untreated BALB/c mice. Figure 8 is a graph depicting the binding ability of anti-ALLY® antibody secreted by a histoincompatible hybridoma cell line grown in mice treated with an immunosuppressive anti-CD4-related antibody. Figure 9 is a graph comparing the binding ability of cyclophilin antibody secreted by a histoincompatible hybridoma cell line grown in mice treated with an immunosuppressive anti-CD4-related antibody with the binding ability of cyclophilin antibody secreted by a histoincompatible cell line grown in untreated mice.

DETAILED DESCRIPTION OF THE INVENTION A majority of T-helper lymphocytes contain a surface differentiation antigen designated CD4 which is commonly referred to as L3T4 in the murine system. This differentiation antigen is a glycoprotein of apparent molecular weight 52,000 as described by Dialynas et al. in J. Immunol., 131:2445-2451 (1983). It appears to be analogous to the Leu3 or T4 differentiation antigen on human helper T cells.

The CD4 molecule is found on the surfaces of all T-helper cells in all species of mammals studied thus far. (Clark et al., Proc. Natl. Acad. Sci. (USA) 84:1649 (1987). It plays an important role in the recognition of foreign molecules by the immune system.

This invention concerns improving ascites production of monoclonal antibodies by immunosuppressing a host in which histoincompatible hybridoma cells are grown by administering an immunsuppressive amount of at least one immunosuppressive anti-CD4-related antibody which interferes with the immune response by binding to host cells involved in the immune response. Thus, these cells are prevented from rejecting the histoincompatible hybridoma cells.

At least one immunosuppressive anti-CD4- related antibody which is capable of down-regulating the functioning of the CD4 molecule and/or the cell or cells with which CD4 is associated can be used to practice the invention. These antibodies can bind to the CD4 molecule itself or to a CD4-reactive substance.

The term CD4-reactive substance means any molecule or cell which is capable of down-regulating the functioning of the CD4 molecule itself or the cell or cells with which CD4 is associated.

.An example of a hybridoma secreting anti-CD4- related monoclonal antibodies is the GK1.5 cell line which was obtained from a fusion of a mouse nonsecretor myeloma SP2/0 with spleen cells from a rat that had been injected with a cloned T cell line as described by Dialynas et al. in J. Immunol., 131:2445-2451 (1983). The monoclonal antibodies secreted are rat IgG 2b antibodies specific against L3T4. Hybridoma cell line GK1.5 is available from the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, MD 20852 under ATCC No. TIB207.

Immunosuppressive anti-CD4-related monoclonal antibodies can be generated de novo using conventional hybridoma technology as mentioned above. Immune lymphocytes can be obtained from any mammalian species. Immunization protocols can be performed either in vivo or in vitro. See, for example, Monoclonal Hybridoma .Antibodies: Techniques and Applications, Hurrell (ed.), (1982) or Monoclonal Antibody Production Techniques and Applications, Schook (ed.). Immunology Series Vol. 33 (1987) .

It is preferred that the immunosuppressive antibodies be grown in a serum free tissue culture medium. This allows the hybridomas to grow. It also makes it easier to purify the resulting antibodies using ammonium sulfate precipitation.

Precipitate obtained in this manner is redissolved in about five to about ten mL of 0.15 molar phosphate buffered saline (PBS), pH of about 7.4, or some other comparable buffer, and dialyzed against the buffer used. Protein concentration in the buffer is estimated by measuring the optical density on a spectrophotometer at a wavelength of about 280 nm using 1.4 as the coefficient of extinction, or by some other standard method of calculating protein concentraiton such as Lowry's technique.

Alternatively, hybridoma cells secreting immunosuppressive anti-CD4-related monoclonal antibodies can be grown in serum containing medium, or as an ascites tumor according to techniques well known to those skilled in the art.

Antibodies produced in this manner are concentrated using techniques such as column chromatography using a column consisting of Protein A obtained from Staphylococcus bound to Sepharose beads (Ey et al., Immunochemistry 15: 429 (1978), or ion exchange chromatography (Menozzi et al., J. Immunological Methods 909: 229-233 (1987).

The term "host" means any animal, such as a rat, mouse, rabbit, etc., in which hybridoma cells can be grown.

The term "antibody" as used herein refers to a monoclonal antibody or a mixture of monoclonal antibodies as well as bispecific antibodies, immunoreactive antibody fragments, and polyclonal antibodies. .An immunoreactive antibody fragment means that fragment which contains the binding region of the antibody. Such fragments may be Fab-type fragments which are defined as fragments devoid of the Fc portion, e.g.. Fab, Fab', and F(ab')₂ fragments, or may be so- called "half-molecule" fragments obtained by reductive cleavage of the disulfide bonds connecting the heavy chain components of the intact antibody.

The GK1.5 hybridoma cell line described above is the preferred source of anti-CD4-related antibodies for practicing the invention.

According to the process of this invention, the host in which histoincompatible cells are to be grown as an ascites tumor are injected intraperitoneally (i.p.) or intravenously with an immunosuppressive amount, e.g., about 100 to about 400 μg, of an immunosuppressive anti-CD4-related antibody which will bind to host cells involved in the immune response prior to injection of the histoincompatible hybridoma cells.

Once the immunosuppressive antibody or antibodies have been administered and after the histoincompatible hybridoma cells are injected, the animals are then monitored and/or weighed to determine whether ascites fluid is accumulating. When the animals have increased in size to the point where the increase can be viewed, an 18 gauge needle is inserted into the peritoneal cavity and the ascites fluid is drained into glass collecting tubes.

Ascites fluid can also be recovered by anesthesizing or killing the animal, making an incision into the abdomen and aspirating the fluid from the peritoneal cavity. It should be clear to those skilled in the art that ascites fluid can be collected using any available technique.

The amount of ascites collected is measured and recorded.

A comparison of the amount of ascites fluid collected from animals which have not been treated with at least one immunosuppressive antibody with those which have been treated with at least one immunosuppressive antibody or a comparison in the weight gained in both groups demonstrates the effect that a particular immunosuppressive antibody or mixture of immunosuppressive antibodies has on ascites production. As the results show below, there is a substantial improvement using the process of the invention.

An immunosuppressive anti-CD4-related antibody as described above can be used alone or in conjunction with a non-antibody-containing immunosuppressive compound such as N,N-bis(2-chloroethyl)tetra-hydro-2H- l,3,2-oxazaphosphorin-2-amine-2-oxide which is commercially available under the trade name cyclophosphamide, or with other immunosuppressive treatments such as sublethal irradiation.

The following examples illustrate the invention:

Example 1

A. Enhanced Ascites Production of an Anti-cyclophilin Monoclonal .Antibody Secreted by a Histoincompatible hybridoma grown in BALB/c Mice Treated With an Immunosuppressive Antibody DBA/IJ mice were immunized with cyclophilin according to a conventional procedure using Freund's adjuvant. This procedure was similar to that described by Krco et al., in J. Immunogenetics 12:197-211 (1985). Anti-cyclophilin monoclonal antibodies were generated by fusing immune lymphocytes obtained from these DBA/IJ mice with the myeloma cell line P3X63-Ag8.653 (P3) of BALB/c mice obtained from the ATCC under identification number ATCC CRL 1580. Cells were fused chemically using PEG 1500 in a manner similar to that described by Kohler and Milstein in Nature 256:495-497 (August 7, 1975).

The histoincompatible hybridomas were grown in a standard cell culture medium containing as its basal medium Iscove's modification of Dulbecco's modified Eagles medium along with approximately 10% fetal bovine serum, 2.4 millimolar L-glutamine, 200 nanomolar beta- mercaptoethanol, 5 micromolar hypoxanthine and 8.8 micromolar thymidine. Cultures were maintained in a humidified incubator at 37° C with 8% CO2.

Approximately 100 μg of the immunosuppressive anti-CD4-related monoclonal antibody, GK1.5 was administered to one group containing five BALB/c mice which had been primed twenty days earlier with 0.5 mL of 2,6,10,14-tetramethylpentadecane (pristane) on days designated -1, 0, and +1. Approximately 50 μg of GK1.5 was injected into the mice on day 2. The GK1.5 monoclonal antibody is secreted by a hybridoma which was obtained from the American Type Culture Collection catalog no. TIB207

The GK1.5 cell line was grown in serum free tissue culture medium until the cells reached a density in the range from about 0.5 x 10⁶ to about 3 x 10⁶ cells per mL. The medium contained equal volumes of Dulbecco's Modified Eagle Medium (D-MEM) and Ham's F-12 Nutrient Mixture (Ham's F-12), 5 μg/mL bovine insulin, 30 μg/mL human transferrin, 237 nmol/mL FeCl_3f 2.6 ng/mL sodium selenite, 800 μmol/mL L-glutamine, 300 nmol/mL beta-mercaptoethanol, 20μmol/mL ethanalamine, and 0.5% culture supernatant fluid obtained from the cell line SPL 4.3 (ATCC CRL 10109, 12301 Parklawn Drive, Rockville, MD, 20852) . Alternatively, 0.5% culture supernatant fluid from the cell line RAW 264.7 (ATCC TIB 71) can be substituted for SPL 4.3 culture fluid. Cells and culture supernatant fluid were subsequently centrifuged at approximately 2000 revolutions per minute (rpm) . Supernatant fluid was saved and mixed with

31.3 grams of (NH )₂S0 (ammonium sulfate) per 100 mL of supernatant fluid. This amount of ammonium sulfate was sufficient to precipitate the majority of proteins, including antibody, present in the culture fluid. This solution was continuously mixed at room temperature for approximately 4 hours after which it was centrifuged for an additional 1 hour at 13,000 RPM. The precipitated antibody formed a pellet at the bottom of the centrifuge tube which was redissolved in 5 to 10 mL of 0.15 molar phosphate buffered saline having a pH of 7.4. Subsequently antibody was dialyzed against an excess of dissolving buffer to remove most of the remaining ammonium sulfate with at least 4 changes of buffer. After dialysis, antibody was recovered and filtered through a 0.2 micrometer filter and stored at 4°C.

Protein concentration (mg/mL) was estimated by measuring absorbance at 280 nanometers on a spectrophotometer and dividing the optical density units obtained by 1.4. Approximately, 0.5 x 10⁵ to 1 x 10⁶ histoincompatible hybridoma cells were injected into the first group of mice on day 0.

A second group containing five BALB/c mice served as the control. They were treated in the same manner as the first group except that they did not receive any immunosuppressive antibody.

The animals were observed for signs of abdominal swelling and when observed, the sharp end of an 18 gauge needle was inserted into the peritoneum and the ascites fluid drained into glass graduated tubes. The amount of fluid collected at this t.ime was measured and recorded. Ascites fluid was collected every 2 to 3 days until either the animal died or stopped producing ascites fluid.

Figure 1 is a graph depicting the total amount of ascites fluid collected from BALB/c mice treated with at least one immunosuppressive anti-CD4-related antibody and the total amount of ascites fluid collected from the mice which were not treated with any immunosuppressive antibody. The results show a substantial increase in the amount of ascites fluid collected from the mice treated with an immunosuppressive anti-CD4-related antibody.

B. Enhanced Ascites Production of Anti-ALLY® Monoclonal Antibody Secreted by a Histoincompatible 17

than the volume -of ascites fluid obtained from untreated mice.

Comparative Example 1 Hybridoma 88DD/73.1 is a cell line obtained by fusing immune lymphocytes from the DBA/IJ mouse strain which had been immunized with cyclophilin as described above in Example 1A with the myeloma cell line P3 obtained from BALB/c mice. The fusion protocol was the same as that described above in Example 1A. Hybridoma cells were grown and injected into BALB/c mice we described in Example 1A except for the following: five groups containing five BALB/c mice each were injected with pristane 9 days prior to the injection of 1 X 10⁶ hybridoma cells. . Mice either received no antibody treatment or were injected with 100 μg i.p. per day on days designated as -1, 0, +1, and +2 with an immunosuppressive antibody obtained from the following cell lines: GK1.5 (ATCC # TIB207) which produces anti-CD4- related monoclonal antibodies; 3.155 (ATCC # TIB211) which produces anti-Lyt 2 antibodies; 2.43 (ATCC # TIB210) which produces anti-Lyt 2.2 antibodies; and/or

7D4 (ATCC # CRL1698) which produces anti-IL-2 receptor antibodies. Another group of mice were treated with 200 μg i.p. per day on days -1, 0, +1, and +2 with anti-la monoclonal antibodies secreted by the hybridoma cell line designated MS/114.15.2 (ATCC #TIB120) . All cell lines producing monoclonal antibodies used for treatment were grown as described for the GK1.5 cell line in Example 1A. Hybridoma cells were, in all cases, injected on day 0. Ascites fluid was collected as described in Example 1A. The amount collected under the different treatment conditions is shown in Figure 5.

The results indicate that the volume of ascites fluid obtained from mice which had been treated with anti-CD4-related antibody GK1.5 produced substantially higher volumes of ascites fluid than did either untreated mice or mice treated with other monoclonal antibodies having immunosuppressive potential.

Example 2 Enhanced Ascites of an Anti-ALLY® Monoclonal Antibody Secreted by a Histoincompatible Hybridoma Grown in BALB/c Mice Treated with an Immunosuppressive Antibody and an Immunosuppressive Drug

The hybridoma cell line 47P/27 used is the same as that described above in Example 1C.

These hybridoma cells were grown and injected into BALB/c mice as described in Example 1A except for the following: animals were injected with pristane 11 to 15 days prior to the injection of 3.8 x 10⁶ hybridoma cells. In addition, mice either received no antibody treatment or were injected with 100 μg i.p. of the immunosuppressive anti-CD4-related antibody GK1.5, on days designated as -1, 0, +1 and +3, where hybridoma cells were injected on day 0. Two groups containing five BALB/c mice each were injected i.p. on day-9 with 2.5 mg of N,N-Bis(2-Chloroethyl)tetra-hydro^H-^S^- oxazaphosphorin^-amine 2 oxide, (SIGMA under the trade name cyclophosphamide) . One group was injected with both cyclophosphamide and the GK1.5 immunosuppressive antibody. The other group received only cyclophosphamide. A third group of mice were treated as described above except they received no treatment with the GK1.5 immunosuppressive antibody. 15

Hybridoma Grown in BALB/c and Fl Mice Treated with an Immunosuppressive Antibody.

DBA/2J mice were immunized with the herbicide ALLY® conjugated to a carrier protein such as Keyhole Limpet Hemocyanin (KLH) or to ovalbumin (OVA) using techniques well known to those skilled in the art. In the case of hybridoma cell line 47B/1.2, ALLY® was conjugated to OVA. The immunization protocol was similar to that described above in Example 1A. The herbicide ALLY® is a selective herbicide for broad leaf weed control in wheat containing the active ingredient metasulfuronmethyl.

Hybridoma 47B/1.2 is a cell line obtained by fusing immune lymphocytes from the DBA/2J mouse strain with the myeloma cell line P3 obtained from BALB/c mice. The fusion protocol was the same as that described above in Example 1A. Histoincompatible hybridoma cells were grown and injected into one group containing five BALB/c mice and one group containing five Fl mice as described in Example 1A except for the following: animals were injected with pristane 11 to 15 days prior to injection of 3.4 X 10⁶ histoincompatible hybridoma cells. The Fl mice were offspring of C57BL/6J strain and DBA/2J strain. The Fl strain is called B6D2Fl/CrlBr and is commercially available from Charles River Laboratories. Each group of mice except the control group was injected with 100 μg i.p. of the immunosuppressive anti-CD4-related antibody GK1.5, on days designated as - 1, 0. +1 and +3. The histoincompatible hybridoma cells were injected on day 0. Ascites fluid was collected as described above.

Figure 2 is a graph depicting the total amount of ascites fluid collected from BALB/c mice treated with at least one immunosuppressive anti-CD4-related antibody 16

and the total amount of ascites fluid collected from untreated mice.

Figure 3 is a graph depicting the total amount of ascites fluid collected from Fl mice treated with at least one immunosuppressive anti-CD4-related antibody and the total amount of ascites fluid collected from untreated mice.

The results for both Figures show an increase in amount of ascites fluid collected from the mice treated with an immunosuppressive antibody.

C. Enhanced Ascites Production of an Anti-ALLY®

Monoclonal .Antibody Secreted by a Histoincompatible Hybridoma Grown in Fl Mice Treated with an Immunosuppressive Antibody.

Hybridoma 47P/27 is a cell line obtained by fusing immune lymphocytes from the LP/J mouse strain which had been immunized with ALLY® conjugated to KLH as described above in Example IB with the myeloma cell line P3 obtained from BALB/c mice. Hybridoma cells were grown and injected into (C57BL/6J x DBA/2J) Fl mice as described above in Example IB except for the following: animals were injected with pristane 11 to 15 days prior to the injection of 1.5 x 10⁶ hybridoma cells. In addition, mice either received no antibody treatment or were injected with 100 μg i.p. of the immunosuppressive anti-CD4-related antibody GK1.5, on days designated as - 1, 0, +1, and +3 with hybridoma cells being injected on day 0. Ascites fluid was collected as described in Example 1A. The amount collected under the different treatment conditions is shown in Figure .

The results indicate that the volume of ascites fluid obtained from mice which had been treated with an immunosuppressive anti-CD4-related antibody produced substantially higher volumes of ascites fluid Ascites fluid was collected as described in Example 1A above and the amount collected under the different treatment conditions is shown in Figure 6.

The results show that the mice treated with an immunosuppressive anti-CD4-related antibody or a combination of such an immunosuppressive antibody and an immunosuppressive drug produced substantially greater amounts of ascites fluid than did the mice which received no treatment.

Example 3

Enhanced Ascites Production of an Anti-Cyclophilin Monoclonal .Antibody Secreted by a Histoincompatible Hybridoma Grown in BALB/c Mice Treated with an Immunosuppresssive Antibody.

Hybridoma cell line 88GG-44.1.1 was used. It was prepared as described above in Example 1.

Eight groups containing five BALB/c mice each which had been injected 8 days prior i.p. with 0.5 mL of Freund's Incomplete Adjuvant (SIGMA Chemical Company) ^•were injected i.p. with 1 x 10⁸ hybridoma cells and received one i.p. injection of 300 μg of the immunosuppressive anti-CD4-related monoclonal antibody GK1.5 on days designated 0, 4, 6, 8, 10, 13, 17, or 21 days prior to being injected with hybridoma cells.

Cells were injected on day 0. Another group containing five BALB/c mice were treated exactly as just described except that they received no GK1.5 antibody treatment. Ascites fluid was obtained from mice as described in Example 1. The total amount of ascites fluid obtained from GK1.5 treated and untreated mice is shown in Figure 7.

The results show that mice which were treated with an immunosuppressive anti-CD4-related antibody as long as ten days prior to injecting the histoincompatible hybridoma cells produced a substantial increase in the amount of ascites fluid collected.

Example 4 A. Ascites Production of Anti-ALLY® Monoclonal Antibody in Fl Mice Treated with an Immunosuppressive Antibody.

Ascites fluid from GK1.5 treated Fl mice injected with the hybridoma cell line 47P/27 as described in Example IC above was tested in an enzyme linked immunosorbent assay (ELISA) for the relative amount of anti-ALLY® monoclonal antibody. This assay was performed using a standard ELISA procedure in which the small chemical herbicide ALLY® was conjugated to the large protein molecule chicken ovalbumin (ALLY®-OVA) , diluted to a concentration of about 50 μg/mL in phosphate buffered saline (PBS) and then dispensed into the wells of a 96 well polystyrene plate. After incubating overnight at 4°C these plates, as well as plates to which ALLY®-0VA had not been added, were washed 3 to 4 times with PBS to remove any ALLY®-OVA that had not adhered to the plastic wells. Subsequent to washing, a solution of 3% bovine serum albumin dissolved in PBS (PBS-BSA) was added to all wells of the ELISA plates so as to cover any exposed plastic to which ALLY®-0VA had not adhered, thereby preventing any nonspecific sticking of reagents used during subsequent steps of the assay.

Plates were incubated for an additional 2 hours at room temperature after which they were washed twice with PBS. Ascites fluid obtained from Fl mice treated with the immunosuppressive anti-CD4-related antibody, GK1.5, and injected with the histoincompatible hybridoma cell line 47P/27 was diluted in PBS-BSA and added to both ALLY®-OVA coated and uncoated plates. These plates were incubated for an additional 2 hours at 37°C after which they were washed 9 times with PBS. Rabbit anti-serum specific for mouse IgG immunoglobulins and conjugated to horseradish peroxidase (HRP) (Zymed) was added to these plates. The plates were washed 12 times with PBS after incubation at room temperature for another hour. One hundred microliters of the substrate (2.2'-azino-di[3-ethyl-benzthiazoline sulfonate(6) ] (.ABTS) was added to each well. The amount of the colored product was measured optically using a monochromatic light source having a wavelength of 405 nanometers. Plates were incubated for 20 minutes at room temperature and the optical density of the fluid in each well was measured using a V-max Plate Reader (Molecular Devices Corporation) . The results of this assay are depicted in Figure 8.

Figure 8 shows that even at a 1 to 78,125 dilution of ascites fluid, antibody present therein bound to ALLY®-OVA coated plates, but not to uncoated plates. Thus, treatment of mice with the immunosuppressive anti-CD4-related antibody GK1.5 did not inhibit the production of Anti-ALLY® monoclonal antibody.

B. Ascites Production of Anti-Cyclophilin Monoclonal Antibody BALB/c Mice Treated with an Immunosuppressive Antibody

Hybridoma cell line 88C-11.6.1 was used. It was prepared as described above in Example 1. Histoincompatible hybridoma cells secreting anti- cyclophilin monoclonal antibody (88CC-11.6.1) were injected into one group containing five BALB/c mice which were treated with antibody GK1.5 as described in Example 1. Eighty-eight mL of ascites fluid was obtained from the 5 GK1.5 treated mice. Fifteen mL of ascites fluid was obtained from the 5 untreated mice. The relative amount of antibody that binds specifically to the protein cyclophilin was determined using the standard ELISA procedure described above in Example 3. The only differences between that assay and the one used in this example are that instead of coating 96 well polystyrene plates with ALLY®-OVA, plates were coated with cyclophilin, and ascites fluid in this assay was serially diluted from 1:50 to 1:8,857,350. The results of this assay are provided in Figure 9.

These results show that the reactivity of the anti-cyclophilin monoclonal antibody was not affected by the immunosuppressive antibody treatment. Antibody activity was detected in ascites fluid obtained from both treated and untreated mice at dilutions approaching one million.

Claims

What is claimed is :

1. A process for improving ascites production of monoclonal antibodies which comprises immunosuppressing a host in whcih allogeneic histoincompatible hybridoma cells are grown by administering an immunosuppressive amount of at lease one immunosuppressive anti-CD4-related antibody.

2 . A process according to claim 1 wherein the immunosuppressive antibody is a monoclonal antibody .

3. A process according to claim 2 wherein the monoclonal antibody is GK1.5.

4. A process according to claim 2 wherein the immunosuppressive antibody is a bispecific antibody.

5. A process according to claim 1 or 2 wherein the immunosuppressive antibody is an immunoreactive antibody fragment.

6. A process according to claim 1 wherein the immunosuppressive antibody is administered in conjunction with an immunosuppressive agent.

7. A process according to claim 6 wherein the immunosuppressive agent is N,N-bis(2-chloroethyl)- tetrahydro-2H-l,3,2-oxazaophosphorin-2-amine-2-oxide.

8. A process according to claim 1 wherein the immunosuppressive antibody is a polyclonal antibody, 24

9. A process according to claim 1 wherein the immunosuppressive antibody is administered in conjunction with sublethal irradiation.