CA2022146A1 - Immunosuppressive monoclonal antibodies, hybridomas and methods of transplantation - Google Patents

Abstract

IMMUNOSUPPRESSIVE MONOCLONAL ANTIBODIES, HYBRIDOMAS AND METHODS OF TRANSPLANTATION Abstract New monoclonal antibodies have been found to have specific binding affinity for the CD3-T cell antigen receptor complex and not to be reactive with human monocytes. The antibodies are immunosuppressive and useful in the treatment or prophylaxis of transplant rejection or GVHD without resulting in acute reactionsor EBV-associated lymphoproliferative syndrome. Representative embodiments include the BC3 monoclonal antibody and the BC3 hybridoma for producing the antibody.

Description

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IMMUNOSUPPRESSIVE MONOCLONAL ANTIBODIES, H~BRIDOMAS
AND METHODS OF TRANSPLANTATION

This invention relates to new monoclonal antibodies having specificity for 5 the CD3-T cell antigen receptor complex, to hybridomas for producing the monoclonal antibodies, and to therapeutic methods and compositions employing the antibodies.

Recipients of allogeneic organ or tissue transplants are routinely treated 10 with immunosuppressive agents to prevent graft rejection and, in the case of bone marrow transplantation, to prevent immunocompetent Iymphoid cells from causing graft-versus-host disease (GVHD3. Drugs used to control rejection or GVHD, such as corticosteroids, azathioprine, methotrexate, cyclosporine and antithymocyte globulin, have been shown to have broadly suppressive effects on cell mediated 15 immune function. However, despite improvements in tissue matching by HLA
typing and presently available improved immunosuppressive agents, acute rejection of the allograft or GVHD remain major impediments to the success of clinical transplantation. In addition, viral infections represent a significant cause of morbidity and mortality in patients immunosuppressed after transplantation.
20 Infections with cytomegalovirus, herpes simplex virus and varicella-zoster virus result primarily from the reactivation of latent virus, although de novo infections have also been documented. Epstein-Barr virus-associated Iymphoproliferative syndromes (EBV-LPS) have been found to occur in as high as five per cent of renal allograft recipients, and 20 per cent of cardiac allograft recipients. Latent EBV
25 infection, together with impaired immune responses, chronic antigenic stimulation by the allograft and direct oncogenic effects of immunosuppressive drugs may represent factors that increase the incidence of EBV-related disease after organgrafting. Severe GVHD, immunosuppressive treatment, HLA incompatibility between donor and recipient and T cell depletion of the donor marrow have been ::- . : .

suggested as possible risk factors for the incidence of EBV-LPS in bone marrow transplant recipients.
In the search for improved immunosuppressive therapy, the use of antilymphocyte antibodies has been previously investigated. Since thymus derived5 lymphocytes (T cells) play a major role in the immune response to allografted tissues and marrow, treatment of transplant recipients with anti-T cell antibodies has been shown to represent a potential method of advancing immunosuppressive therapy. The administration of antithymocyte globulin (ATG) to transplant recipients has been employed for some time. More recently, advances in 10 monoclonal antibody technology have permitted the development of monoclonal antibodies specific for the CD3-T cell receptor complex to be used for this purpose. For example, Cosimi et al., The New England Joumal of Medicine, Vol. 305, No. 6, pp. 308-314, August 6, 1981, and the Ortho Multi-Center Transplant Study Group, The New England Joumal of Medicine, Vol. 313, No. 6, 15 pp. 337-342, August 8, 1985, disclose the use of monoclonal antibody OKT3 in the treatment of renal transplant patients. OKT3 is a murine monoclonal antibody of IgG2a isotype specific for the CD3-T cell antigen receptor complex (see U.S. Patents Nos. 4,361,549, 4,515,893, and 4,654,210). Martin et al., Transplantation Proceedings, Vol. XVI, No. 6, pp. 1494-1495, 1984, and 20 Remlinger et al., Human Immunology, Vol. 9, pp. 21-35 (1984) disclose the use of murine IgG2a monoclonal antibodies 9.6 (anti-CD2), 10.2 (anti-CD5), 12.1 (anti-CD6), 35.1 (anti-CD2), and 64.1 (anti-CD3) in the treatment of acute graft-versus-host disease. However, the in vivo use of anti-CD3 murine IgG2a monoclonal antibodies, including OKT3, has been associated with acute side effects, including 25 pyrexia, chills, dyspnea, chest pain, vomiting, wheezing, nausea, diarrhea, tremor, and increased susceptibility to cytomegalovirus and herpes simplex infection. Inaddition, the use of murine IgG2a anti-CD3 monoclonal antibodies, specifically antibody 64.1, as an immunosuppressive agent for the treatment of acute GVHD
has been documented to be associated with a substantial risk of EBV-associated 30 lymphoproliferative disorders (see Martin et al., Annals of Intemal Medicine,Vol. 101, pp. 310-315, 1984 and Martin et al., American Journal of Kidney Diseases, Vol. XI, No. 2, pp. 149-152, Feb. 1988. EBV-associated lymphoproliferative disorders have similarly been associated with administrationof the OKT3 antibody.
The EBV-related lymphoproliferative syndrome has emerged as a major complication in attempts to treat graft rejection and acute GVHI3 with monoclonal antibodies. Despite the advances that have been made in , :: : :. . . ,-: - , :.. ~ . :
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immunosuppression therapy with monoclonal antibodies, particularly with the IgG2a anti-CD3 monoclonal antibodies OKT3 and 64.1, a major need exists for improved monoclonal antibodies which are effective as immunosuppressive agents, but which are not associated with acute toxicity and do not enhance the risk of 5 B-cell proliferation and transformation.
It is accordingly one object of this invention to provide hybridomas which produce monoclonal antibodies specific for the human CD3-T cell antigen receptorcomplex.
It is a further object of the invention to provide monoclonal antibodies l0 specific for the CD3-T antigen receptor complex which are not associated withacute toxicity and do not increase the risk of EBV-associated lymphoproliferative syndrome when administered in vivo.
It is a still further object of the invention to provide methods and therapeutic compositions for the treatment or prophylaxis of transplant rejection l 5 and graft-versus-host disease.
Other objects and advantages of the invention will become apparent from an examination of the present disclosure.

There have now been discovered novel hybridomas which are capable of 20 producing monoclonal antibodies specific for the CD3 differentiation cluster of human T lymphocytes. The antibodies are effective as immunosuppressive agents when administered in vivo, but are not pyrogenic, do not induce respiratory distress, and do not increase the risk of EBV-related lymphoproliferative syndrome. Accordingly, the antibodies are effective in the treatment or 25 prophylaxis of organ or bone marrow rejection by a transplant recipient, in the treatment or prophylaxis of graft-versus-host disease, or for diagnostics purposes.
Preferred antibodies of the invention are murine monoclonal antibodies of the IgG2b isotype which do not interact with Fc receptors on human monocytes.
A representative embodiment of the invention is the hybridoma designated BC3 30 which produces a monoclonal antibody, also designated BC3.
The hybridoma BC3 has been deposited with the American Type Culture Collection, 12301 Park Lawn Drive, Rockville, Maryland 20852, and assigned the ATCC accession No. HB 10166.

FIGURE 1 is a graphic representation of the comodulation of the epitopes on peripheral blood lymphocytes bound by either antibody 6~.1 or antibody BC3, demonstrated by staining with either FITC-conjugated antibody BC3 or FITC-conjugated antibody 6~.1. respectively.

2022~fi FIGURE 2 is a graphic representation of cell proliferation of peripheral blood mononuclear cells induced by antibody BC3 or antibody 64.1, as measured bythymidine incorporation assay.
FIGURE 3 is a graphic representation of cell proliferation of peripheral 5 blood mononuclear cells induced by an alloantigen in the presence of antibody BC3, as measured by thymidine incorporation assay.

As used herein, the term "monoclonal antibody" means an antibody composition having a homogenous antibody population. It is not intended to be l0 limited as to the source of the antibody or the manner in which it is made.
As used herein with respect to the exemplified murine monoclonal anti-CD3 antibodies, the term "functional equivalent" means a monoclonal antibody having selective binding affinity for the CD3-T antigen receptor complex of human T
lymphocytes (T cells) and which does not bind to human monocyte Fc receptors.
As used herein with respect to the exemplified hybridomas of the invention, the term "progeny" is intended to include all derivatives, issue, and offspring of the parent hybridoma that produce the monoclonal antibody of the parent or a functional equivalent thereof, regardless of generation of karyotic identity.
Antibodies of the invention specifically bind the Tpl9-29 antigen of the CD3 20 differentiation cluster of human T lymphocytes as defined in Bernard et al. (Eds.), in Joint Report of the First International Workshop on Leukocyte DifferentiationAntigens, New York, pp. 9-142 (Springer-Verlag, 1984). Unlike well-known anti-CD3 monoclonal antibodies OKT3 and 64.1, however, the antibodies of the invention are of the IgG2b isotype and do not interact with Fc receptors on human 25 monocytes (see Kenneth et al., European Journal of Immunology, Vol. 16, pp. 478-486, 1986). Accordingly, the in vivo administration of the monoclonal antibodies of the invention does not result in the inducement of EBV-related lymphoproliferative syndrome, febrile reactions, bronchospasm or hypotension typically present after administration of the IgG2a anti-CD3 monoclonal 30 antibodies OKT3 and 64.1, complications which are believed to result from thesecretion of lymphokines after antibody binding to T cells crosslinks to Fc receptor position accessory cells.
A presently particularly preferred, representative embodiment of the antibodies of the invention is the antibody BC3 produced by the BC3 hybridoma 35 (ATCC No. HB 10166) and functional equivalents thereof.
Antibody-producing lymphocytes used as fusion partners to make the hybridomas of the invention may be generated by immunizing mice, such as , ~

BALB/c mice, with PHA activated T cells or membrane extracts made therefrom. The mice are preferably innoculated intraperitoneally with an immunogenic amount of the cells or extract and then preferably boosted with similar amounts of the immunogen over a period of about 10 days to two weeks.
5 Spleens may be collected from the immunized mice after the final booster injection and processed to obtain a cell suspension for use in fusion with myeloma cells.
Myeloma cells used as fusion partners to make the hybridomas of the invention are well known and readily available in the art. In a representative l0 embodiment, more fully described herein, BALB/c MOPC21 NS1/1 is used as the myeloma fusion partner, although other myeloma cell lines may be used for this purpose.
In producing the hybridomas of the invention, the spleen cells from immunized mice are combined with the myeloma cell fusion partners in the l5 presence of a suitable fusion agent, such as polyethylene glycol. After fusion, the cells are separated from the fusion medium and grown in a selective medium, suchas HAT medium, to eliminate nonhybridized parent cells. Supernates from the remaining hybridomas are screened for specific binding affinity for the CD3-T cell antigen receptor complex of human peripheral T cells and lack of binding affinity 20 for human monocytes by conventional assay procedures, such as by radioimmunoassay, enzyme immunoassay, fluorescence immunoassay, or the like.
In a representative embodiment, hybridomas of interest may be selected by screening supernates for blocking the binding of a fluorescence in isothiocyanate (FITC) conjugated anti-CD3 antibody by using flow microfluorimetry.
25 Specifically, 0.5 x 106 peripheral blood mononuclear cells obtained from volunteers and purified by density gradient centrifugation on Ficoll Hypaque (S.g. 1077) may be incubated with 50u1 of hybridoma culture supernates for 30 min at 4C in the presence of 0.1% sodium azide. Cells may then be incubated with saturating concentration of a known CD3-specific antibody conjugated to FITC.
30 Incubation may be carried out for 30 min. at 4C in the presence of 0.1% sodium azide. After washing, cells may be evaluated by flow microfluorimetry. Green fluorescence intensity of samples preincubated with hybridoma supernates may be compared to green fluorescence intensity of samples preincubated with medium.
Hybridoma supernates capable of reducing the fluorescence intensity of cells 35 stained with FlTC-conjugated CD3 antibody should contain an antibody specific for the CD3 antigen.

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Supernates found to contain a CD3 antibody may be further screened for the presence of antibodies of the desired IgG2b isotype as follows. 50 ul of hybridoma supernate containing the newly found anti-CD3 antibody may be incubated with 0.5 x 106 unfractionated peripheral blood mononuclear cells in 200 ul round bottom 5 well plates for 72 hours at 37C in the presence of 5% CO2 atmosphere. Induction of DNA synthesis may be measured by incorporation of tritiated thymidine. Since antibodies of IgG2a, IgG1 and IgG3 isotype are known to be mitogenic for unfractionated peripheral blood mononuclear cells, supernates containing antibodies inducing DNA synthesis are discarded. Supernates containing anti-CD3 l0 antibodies not inducing DNA synthesis may contain antibodies of the IgG2b or the IgM isotype. Anti-CD3 antibodies of the IgG2b isotype may then be identified by various conventional means known in the art, such as by ELISA (enzyme-linked immunosorbent assay) or double immunodiffusion, using antibodies or antisera specific for IgG2b antibodies.
l 5 Hybridomas determined by a suitable screening procedure to produce antibodies of the invention may be grown in vitro in culture medium or in vivo, preferably in the peritoneal cavity of a mouse, using conventional procedures.
Monoclonal antibodies of the invention may be separated from the culture medium, or body fluids, such as ascites fluid or serum, and purified by known 20 immunoglobulin purification procedures, such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography and ultrafiltration.
The foregoing and other aspects of the invention may be better understood in connection with the following examples, which illustrate a presently preferred representative embodiment of the invention.
E~AMPLE 1 - Hybridoma Production Hybridoma BC3 was prepared by fusion of immune murine spleen cells with a murine myeloma cell line, substantially as described by Kohler and Milstein, Nature, Vol. 256, p. 495 et seq., 1975. Specifically, a BALB/c mouse was immunized intraperitoneally with 2 x 106 to 2 x 107 1:1 cell mixture of PHA
activated T cells on day 18, day 11 and day 4, before infusion. Spleen cells of the mouse were fused with HGPRT myeloma cell line BALB/c MOPC21 NS1/1 obtained from Dr. Caesar Milstein (Molecular Research Council, Cambridge, England). Selected hybrid cells were subcultured at low density, to~ether with a"feeder cell" suspension of thymocytes from BALB/c mice. These cell mixtures contained hybrid cells at a concentration of 2.5 x 101/ml and thymocytes at a concentration of 4 x 106/m9. A 200 I-l volume of this suspension was dispensed into microwells, resulting in a seeding of 5 hybrid cells/well. Hybridoma culture , .'': :
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7 20221~6 supernates were screened, as described above, for anti-CD3 specificity and IgG2bisotype, and cloning of cells from a positive well was performed by limiting dilution (100 hybridoma cells/300 wells) with thymocyte feeder cells as described above, to obtain BC3 hybridoma cells. The hybridoma cells were cultured in 5 RPMI 1640 containing 159S fetal calf serum, 2 mM added glutamine, 1 mM
pyruvate, 100 units/ml penicillin, and 1 mg/ml streptomycin. Cultures were maintained at 37C in a humidified atmosphere containing 5% CO2. Cell density was maintained between 0.1 x 106/ml and 0.4 x 106/ml by splitting the cultures every two to three days.
l O EXAMPLE 2 - BC3 Ascites Fluid Production BALB/c mice were pretreated with 0.5 ml of intraperitoneal pristane (Alderidge, Milwaukee, Wl), and then innoculated by intraperitoneal injection of 5-10 x 106 BC3 hybridoma cells prepared in accordance with Example 1. After five to 15 days, ascites fluid was obtained by insertion of an 18 gauge needle into the 15 peritoneum and collection by open draining into a sterile 5 ml tube. Fluids were allowed to clot at room temperature for one-half hour, and then centrifuged at 2,500 x G for 10 minutes at 4C. Multiple ampules of hybridoma cells from ascites fluid of individual selected mice were cryo-preserved in 50% RPMI 1640 medium and 10% DMSO.
EXAMPLE 3 - Antibody Purification Ascites fluid obtained according to Example 2 was purified by sequential G-25 Sephadex filtration and affinity chromatography on Protein A-Sepharose CL-4B to obtain purified BC3 monoclonal antibody. All handling of monoclonal antibodies was done with disposable pharmaceutical syringes or other pyrogen-free 25 instruments. Protein A-Sepharose CL-4B columns were stored at 4C in phosphate-buffered saline (PBS) pH 7.2 containing 0.196 Na azide. Columns were purged with 3 bed volumes of PBS before application of ascites fluid. Where possible, pharmaceutical-grade reagents were employed in preparation of buffers. EXAMPLE 4 Antibody Characterization and Class Determination Supernate from the BC3 hybridoma of Example 1 contained a murine IgG2b/k antibody, designated BC3, as determined by an ELISA assay using peroxidase-conjugated rabbit anti-mouse Ig isotype specific antibodies.
The immunoprecipitate of 125I surface labeled human PBL lysate with antibody BC3 consisted of a protein complex with a molecular weight of 19-29 kDa by one-dimensional SDS-PAGE. The specificity of BC3 for the CD3 complex was demonstrated by comodulation experiment~ with the known CD3 antibody - ..- . ., : .

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202~1~6 64.1. In the comodulation experiments, 5 x 105 peripheral lymphocytes were incubated for 16 hours at 37C with either medium alone or with antibody BC3 and then stained at 4C for 30 min with FlTC-conjugated antibody 64.1, or with antibody 64.1 and stained FlTC-conjugated BC3. The fluorescence intensity of 5 antibody bound to the lymphocytes was then determined. As shown in FIG. 1, the fluorescence intensity associated with lymphocyte-bound FlTC-conjugated antibody BC3 (represented by the medium alone~ is substantially reduced in the presence of antibody 64.1; and, conversely, the fluorescence intensity associated with lymphocyte-bound FlTC-conjugated antibody 64.1 (represented by the l 0 medium alone) is substantially reduced in the presence of antibody BC3. Thisexperiment demonstrated that both the BC3 and 64.1 antibodies bind to the same CD3 molecular complex.
The BC3 and 64.1 antibodies were further evaluated to characterize their mitogenic properties. Five x 104 peripheral blood mononuclear cells were l5 incubated at 37C in a 5% C02 atmosE!here in the presence of 101lg of antibody 64.1, antibody BC3 or antibody 9E8 (an irrelevant antibody control). At various time intervals, cell proliferation was measured by thymidine incorporation assay, as described in Schooley et al., "T-lymphocyte subset interactions in the cell-mediated immune response to Epstein-Barr virus." Cell Immunol., Vol. 86, 20 pp. 402-412, 1984. As shown in FIG. 2, antibody 64.1 induced cell proliferation, which was maximal on days 3 and 4, whereas antibody BC3 did not induce any cell proliferation.
The effect of antibody BC3 on T cell proliferative response was further characterized as follows. Five x 104 peripheral blood mononuclear cells were 25 incubated with an equal number of HLA-class ll, incompatible, irradiated (3000 cGy) peripheral blood mononuclear cells in the presence of 10 ug/ml of antibody BC3 or 9E8 (an irrelevant antibody control). At various time intervals, cell proliferation was measured by thymidine incorporation assay. As shown in FIG. 3,antibody BC3 completely abrogated cellular proliferative response to alloantigens.
E~AMPLE 5 Immunosuppressive Activity and Effect on Lymphoproliferation To evaluate whether BC3 antibody has immunosuppressive properties without the toxicities associated with administration of the other CD3 antibodies, a clinical study was undertaken using escalating doses of BC3 for treatment of 35 patients with acute GVHD refractory to corticosteroids. Ten patients were treated with the antibody. One of 65 infusions was followed by fever and none byother acute reactions. One patient who had been previously treated with ATG

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20~21~6 g subsequently received seven infusions of antibody BC3 for treatment of skin GVHD. After an initial response to BC3 treatment, a second course of ATG was administered for treatment of recurrent disease. Later, this patient developed alethal EBY-LPS. Because of the confounding role of ATG treatment and the other 5 risk factors, namely an HLA-incompatible transplant, immunosuppressive treatment with cyclosporine and corticosteroids and severe acute GVHD, the contributory role played by antibody BC3 in the development of EBV-LPS could not be reliably assessed. None of the other patients developed this complication.
Three patients were not evaluable for clinical response: two did not live l O long enough to complete the treatment course, and one had liver GVHD
superimposed on pretransplant hepatitis and veno occlusive disease. Seven patients were evaluated for response: disease resolved in all seven cases, skin in four, liver in one, and both skin and liver in two. A BC3 antibody dose of 0.1/mg/kg/day was found to be effective in all patients except two, who had the l 5 dose doubled after three days because of persistent GVHD. In one case, the response was not sustained and the patient required subsequent treatment with ATG, as described above. In the other six cases, the response was sustained until death from veno occlusive disease of the liver (one), and CMV pneumonia (two), or indefinitely in the three patients surviving more than 100 days after 20 transplantation. From these data, it is concluded that administration of antibody BC3 is not associated with acute toxicity and that it is effective treatment of GVHD.
When used in vivo for therapy, the immunosuppressive antibodies of the invention are administered to a patient in therapeutically or prophylactically 25 effective amounts, i.e., amounts sufficient to eliminate or reduce T-cell interaction with transplanted organ or bone marrow tissue in a transplant recipient and thereby reduce or eliminate rejection of the organ or bone marrow transplant. The antibodies will typically be administered parenterally, preferably intravenously, but may be administered by other routes known in the art. Suitable 30 dose and dosage regimens will depend upon the desires of the treating physician, the nature, history and medical condition of the patient, and the specific condition being prophylactically or therapeutically treated. Typical therapeutically or prophylactically effective amounts of antibody for intravenous administration will range from about 0.05 mg/kg/day to about 10 mg/kg/day of patient weight for 35 periods of from about two to about 14 days, or longer.
For parenteral administration, antibodies of the invention will be formulated in unit dosages in injectable form in association with a pharmaceutically , " ! ~ ' ' ' '` :'' : ::: ., .... ::,, . . "' "' ', .' -- ~ ' ' '" .

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acceptable parenteral carrier. Suitable, acceptable carriers are typically nontoxic and nontherapeutic. Examples of suitable carriers include saline, Ringer's solution, and human serum albumin solutions. Nonaqueous vehicles, such as fixed oils and ethyloleate and liposomes may also be employed. Suitable vehicles may 5 additionally contain minor amounts of additives, such as substances that enhance isotonacity and chemical stability, e.g., buffers and preservatives. Antibody will typically be present in the pharmaceutical carrier for direct administration at final concentrations of from about 0.05 mg/ml to about 5.0 mg/ml.
Antibodies of the invention may be administered alone, or in conjunction 10 with other immunosuppressive agents, if desired. Other representative immunosuppressive agents suitable for use in conjunction with the antibodies of the invention include the immunosuppressive monoclonal antibodies, corticosteroids, azathioprine, methotrexate, cyclosporine and antithymocyte globulin.
While the invention has been described in connection with certain presently preferred, illustrative embodiments, it is apparent that various modifications, such as use of the antibodies of the invention for diagnostic or other purposes, can be made by one skilled in the art without departing from the spirit and scope ofthe invention. Except as precluded by the prior art, any such modifications are 20 intended to be within the scope of the following claims.

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Claims (16)

1. A hybridoma which produces murine monoclonal antibodies reactive with the CD3-T cell antigen receptor complex of human peripheral T cells and not reactive with the Fc receptors of human monocytes.

2. A hybridoma of Claim 1, which produces IgG2b isotype antibodies.

3. A hybridoma of Claim 2 having the characteristics of hybridoma BC3 (ATCC No. HB 10166).

4. A monoclonal antibody which reacts with the CD3-T cell antigen receptor complex of normal human peripheral T cells, but does not react with the Fc receptors of human monocytes.

5. A monoclonal antibody of Claim 4 having an IgG2b isotype.

6. A monoclonal antibody of Claim 5 having the characteristics of that produced by hybridoma BC3 (ATCC No. HB 10166).

7. A method of producing a murine monoclonal antibody reactive with the CD3-T cell antigen receptor complex of human peripheral T cells, but not reactive with the Fc receptors of human monocytes, comprising culturing the hybridoma BC3 (ATCC No. HB 10166) in a suitable medium and recovering antibody produced 5 by the hybridoma.

8. Monoclonal antibodies produced by the method of Claim 7.

9. A method of producing murine monoclonal antibody reactive with the CD3-T cell antigen receptor complex of human peripheral T cells, but not reactive with human monocytes comprising injecting the hybridoma BC3 (ATCC
No. HB 10166) into the peritoneal cavity of a mouse and recovering antibody fromascites fluid or serum of the mouse.

10. Monoclonal antibodies produced by the method of Claim 9.

11. A therapeutic composition comprising an amount of a murine monoclonal antibody reactive with the CD3-T cell antigen receptor complex of human peripheral T cells and not reactive with human monocytes, effective to reduce or eliminate rejection of an organ or bone marrow transplant by a transplant recipient, or effective to reduce or eliminate graft-versus-host disease (GVHD), together with a pharmaceutically acceptable carrier.

12. A therapeutic composition comprising an amount of a murine monoclonal antibody produced from a hybridoma having the characteristics of BC3 (ATCC No. HB 10166), effective to reduce or eliminate rejection of an organ or abone marrow transplant by a transplant recipient, or effective to reduce or eliminate graft-versus-host disease (GVHD), together with a pharmaceutically acceptable carrier.

13. A method of treatment of an organ or bone marrow transplant recipient comprising administering to the recipient an amount of a murine monoclonal antibody reactive with the CD3-T cell antigen receptor complex of human peripheral T cells and not reactive with human monocytes, effective to cause reduction or elimination of rejection of the transplant by the recipient.

14. The method of Claim 13, wherein the monoclonal antibody is produced from a hybridoma having the characteristics of BC3(ATCC No. HB 10166).

15. A method of treatment of a transplant patient comprising administering to the patient an amount of a murine monoclonal antibody reactive with the CD3-T-cell antigen receptor complex and not reactive with the Fc receptors of human monocytes, effective to reduce or eliminate graft-versus-host disease (GVHD).

16. The method of Claim 15, wherein the monoclonal antibody is produced from a hybridoma having the characteristics of BC3(ATCC No. HB 10166).