MXPA96002569A - Monoclonal antibodies to antigens expressed through hematopoyeti facilitatory cells - Google Patents

Abstract

The present invention relates to monoclonal antibodies (MAb) to haematopoietic facilitator cells (FC). In particular, it relates to MAbs against antigens expressed by murine FC, methods for generating the antibodies, and methods of using same. Marker-directed MAbs that are expressed specifically or at higher levels by FC than by most other marrow cells that have a wide range of applications including but not limited to the rapid isolation of CFs, the identification of CFs in a donor cell preparation, and the molecular cloning of the genes that encode the corresponding reference antigens

Description

"MONOCLONAL ANTIBODIES TO ANTIGENS EXPRESSED BY HEMATOPOYETIC FACILITATORY CELLS" 1. INTRODUCTION The present invention relates to monoclonal antibodies (MAb) to hematopoietic (FC) facilitator cells. In particular, it relates to MAb against antigens expressed by murine FC, methods for generating the antibodies and methods for using same. MAbs targeting markers that are expressed specifically or at higher levels by FC through most other bone marrow cells have a wide range of applications, including but not limited to the rapid isolation of HR, the identification of CF in a preparation of donor cells and molecular cloning of the genes coding for the corresponding reference antigens. 2. BACKGROUND OF THE INVENTION A primary focus in the transplantation of solid organs is the grafting of the donor organ without an ie response to graft rejection, generated by the recipient, while the iocompetence of the recipient against other foreign antigens is conserved. Typically, non-specific non-specific agents such as cyclosporin, ethotrexate, steroids and FK506 are used to prevent host rejection responses. They must be administered on a daily basis and if they are stopped, a rejection of the graft usually results. However, non-specific iosuppressive agents work by suppressing all aspects of the ie response, thereby greatly increasing a recipient's susceptibility to infections and diseases including cancer. Furthermore, despite the use of iosuppressive agents, graft rejection still remains a major source of morbidity and mortality in the transplantation of human organs. Only 50 percent of heart transplants survive 5 years and 20 percent of kidney transplants survive 10 years. (See Powles, 1980, Lancet, page 327, Ramsay, 1982, New Engl. J. Med., Page 392). Most human transplants fail within 10 years without permanent acceptance. Therefore, it would be a considerable advance if tolerance could be induced in the receiver. The only known clinical condition in which tolerance of specific transplantation occurs is the complete systematic donor that occurs reliably and reproducibly when the chimerism is created through bone marrow transplantation. (See Qin et al., 1989, J. Exp. Med., 169: 779, Sykes et al., 1988, Iol. Today 9:23, Sharabi et al., 1989, J. Exp. Med. 169: 493). This has been achieved in neonatal and adult animal models as well as in humans by total lymphoid irradiation of a recipient followed by bone marrow transplantation with the donor cells. The extensive application of bone marrow transplantation to areas outside malignancy has been limited by graft-versus-host disease (GVHD). The success rate of bone marrow transplantation depends, in part, on the ability to closely match the major histocompatibility complex (MHC) of the donor cells with that of the recipient cells. MHC is a complex gene that encodes a large formation of individually unique glycoproteins expressed on the surface of both the donor cells and the host cells that are the main targets of the ie responses of transplant rejection. In the human being, the MHC is referred to as HLA. When HLA identity is achieved by matching a patient with a family member such as a child of the same parents, the likelihood of a satisfactory result is relatively high, even though the GVHD has not yet been completely eliminated. The incidence and severity of GVHD are directly related to the degree of genetic disparity. In fact, only, one or two mismatches of antigen is acceptable because the GVHD is very serious in cases of greater disparities. When allogeneic bone marrow transplantation is carried out between two people who are maladaptive in MHC of the same species, common complications involve graft failure, insufficient immunocompetence and high incidence of GVHD. GVHD is a potentially lethal complication in bone marrow transplantation that occurs in approximately 35 percent to 50 percent of recipients of untreated identical HLA marrow grafts (Martin et al., 1985, Blood 66: 664). ) and up to 80 percent of the recipients of the maladaptive HLA marrow. Unfortunately, only 30 percent of patients usually have a donor who is a member of the family with appropriately adapted HLA-identical, and therefore most patients are excluded from being considered for bone marrow transplantation, or if they carry out the transplant they must tolerate a high risk of GVHC. GVHD results from the ability of mature immunocompetent immune cells (primarily T cells, but some B cells and natural killer cells) in the donor graft to recognize host tissue antigens as foreign and invoke an adverse immune reaction. Mixed allogeneic reconstitution where a mixture of the donor and recipient marrow is transplanted results in improved immunocompetence and increased resistance to GVHD, successful grafting is still not consistently achieved and GVHD still occurs frequently in recent transplant studies. of bone marrow suggest that the main cause of GVHD are T cells, since the removal of T cells from the donor cell preparation is associated with a reduction in the incidence of GVHD. (Vallera et al., 1989, Transplant, 47: 751; Rayfield, 1984, Eur. J. Immunol., Page 308; Vallera, 1982, J. Immunol., 128: 871; Martin and Korngold, 1978, J. Exp. Med., Page 1687; Prentice, 1984, Lancet Page 472). After T cells were implicated to be the predominant mediators of GVHD in animal models, aggressive protocols for T-cell depletion (TCD) of donor bone marrow were instituted. Even though the incidence of GVHD decreased dramatically, TCD was accompanied by a significant increase in graft failure, indicating that T cells might also have a positive role in bone marrow grafting. (Soderling, J. Immunol., 1985, 135: 941, Vallera, 1982, Transplant 33: 243, Pierce, 1989 Transplant., Page 289). The increase in graft failure in human recipients varied from approximately 5 percent to 70 percent of the total patients and was related to the degree of MHC disparity between the donor and recipient (Blazar, 1987, UCLA Sy p., page 382, Filipovich, 1987, Transplant., page 62, Martin et al., 1985, Blood 66: 664, Martin et al., 1988, Adv. Immunol. 40: 379). Failed graft patients usually die even when a second bone marrow transplant is performed. Consequently, the majority of transplant institutions in the United States have abandoned TCD of the donor's bone marrow and, therefore, a high degree of GVHD must be tolerated leading to significant morbidity and mortality. Therefore, the application of bone marrow transplantation as a form of treatment is limited only to environments where the potential of GVHD is clearly counteracted by the potential benefit. Therefore, it was anticipated that administration of purified bone marrow stem cells would lead to optimal grafting and avoid GVHD. However, recent studies have shown that purified bone marrow stem cells are only grafted onto genetically identical receptors but not genetically different receptors. The implication that T cells could participate in both detrimental reactions of GVHD and the facilitation of the useful graft was an enigma that existed for a long time in the scientific community. The researchers began looking for the possible existence of a component of the bone marrow that could facilitate the bone marrow graft but that was removed during the TCD. The identification and purification of this facilitative component would potentially allow the design of transplant protocols to selectively prevent GVHD, while retaining the cells that can improve the graft. Even though most of the researchers speculated that the facilitation component was a haematopoietic cell different from hematopoietic stem cells, this component had never been identified or characterized until recently. In fact, all the evidence pointed to the implication of a form of T cells. It was recently discovered that a population of cells referred to as FC facilitates the grafting of hematopoietic stem cells into a receptor without producing GVHD, and this cell expresses several markers shared by other leukocytes. The identification of specific markers expressed by FC greatly helps the rapid isolation of this type of cell. 3. SUMMARY OF THE INVENTION The present invention relates to MAbs directed to antigens expressed by murine FC, methods for generating the antibodies and methods for using same to isolate FC. The invention is based, in part, on the applicants' discovery that CF plays a critical role in promoting the ability of donor hematopoietic stem cells to graft into a lethally irradiated allogeneic or xenogeneic recipient. Even though the murine FCs are morphologically distinct from all other known cell types and have been known to express Thy-1, CD2, CD3, CD5, CD8, CD45 and MHC class II (on the low or intermediate scale in comparison with B cells and dendritic cells), these markers individually do not readily distinguish CF from other bone marrow cells. Therefore, CF isolation and enrichment now employ an annoying and delayed multiple step procedure involving positive and negative selection.

In order to develop a method for the rapid identification of CFs in a mixture of cells and their subsequent isolation from it, MAbs can be produced to antigens specifically or expressed more selectively by FC than by other cells, assuming that these antigens exist . The generation of MAb requires the use of FC as immunogens, but since CFs are present in natural tissue sources at low amounts (approximately 0.05 percent), it is practically difficult to obtain a high yield from an FC-enriched population to be used in the immunization. Even when whole bone marrow preparation with little or no enrichment for FC can be used as immunogens. It is unlikely that MAb can rise to FC markers since other bone marrow cells are present in much higher numbers and express other highly immunogenic antigens that can dominate antibody responses to molecules associated with FC. In an effort to generate MAb to FC, it is recognized that FCs can share certain antigens from the cell surface with brain tissue as shown by the ability of the rabbit-anti-mouse brain antiserum (RAMB) to reduce the Graft level of the donor bone marrow cell, presumably due to a depletion of FC. Therefore, brain tissue is prepared and used to immunize animals. The fusion of the cells is carried out using spleen cells from immunized animals and the resulting hybridomas are first selected for the secretion of antibodies in their supernatants. Then, MAbs are further selected because of their ability to deplete FC activity in vivo, as manifested by allogeneic chimerism mixed in receptors after reconstitution with donor bone marrow cells treated with the antibodies. The MAbs that exhibit these activities in this selection procedure are selected for further characterization. The invention is described by way of examples where the mouse brain tissue is prepared and used to immunize rats. After several immunizations, the rats are sacrificed and their spleen cells are fused with the mouse myeloma cells. The resulting hybridomas are first selected due to their secretion of rat antibodies from the IgG or IgM isotopes. Positive hybridomas are further tested by reacting their supernatant fluids with bone marrow cells from the donor mouse (H-2 ^) before co-administration with the bone marrow of the TCD donor H-2 ^ to the H-2 ^ recipients. In this model, untreated allogeneic donor bone marrow cells give rise to completely allogeneic chimeras (100 percent), whereas donor cells treated with RAMB or anti-Thy-1 antibody produce low levels of mixed allogeneic chimerism, in case of producing it in receptors, supposedly due to the decrease of FC in the preparation of donor cells. Three MAb, designated R7.6.2, R340.3.1 and R373.6.3 are able to deplete the FC, producing mixed allogeneic chimeras. A wide variety of uses for MAb to antigens expressed by FC are encompassed by the invention described herein, including, but not limited to the identification of FC in a donor cell preparation, the isolation and enrichment of FC from a mixture of cells, and the molecular cloning of the reference antigens. corresponding. 4. BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1A and IB Cells in the bone marrow of the untreated donor produce completely allogeneic chimeras. Only alogeneic cells (H-2 ^) are detected. FIGURES 2A and 2B Bone marrow cells from the donor treated with RAMB produce mixed allogeneic chimeras. Both syngeneic and allogeneic cells are detected. FIGURES 3A and 3B Bone marrow cells from the donor treated with anti-Thyl .2-produce mixed allogeneic chimeras. Both syngeneic and allogeneic cells are detected.

. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to MAb to antigens expressed by murine FC, methods for generating these antibodies and uses of these MAbs. Although the specific methods and methods described herein are exemplified using murine brain tissue to induce rat MAb against mouse FC, they are illustrative only for the practice of the invention. The analogous methods and techniques are equally applicable to a variety of animal hosts immunized against brain tissue, partially purified FC or FC antigens to produce MAb against FC markers, including that expressed by human HR. . 1 PREPARATION OF IMMUNOGENS In order to generate MAb to antigens selectively expressed by FC, there are two main impediments that must be overcome first. The first is related to the low amounts of FC in natural tissues and therefore they need to be enriched to sufficient amounts and in a relatively pure form for use with immunogens. It is estimated that 4000 hours of cell sorting would be required to obtain sufficient numbers of FC purified from the bone marrow for use in the immunization of an animal, if the CFs are purified to a purity of > 95 percent. Even though the FC activity allows the use of these cells in relatively small numbers when they are enriched, it is preferred that they be enriched until > 50 percent to be used as immunogens. FC can be isolated from any of the tissues in which they reside, using a variety of separation methods. In accordance with this aspect of the invention, human HR can be isolated from the bone marrow. The procedures involving repetitive density gradient centrifugation, positive selection, negative selection or combination thereof can of course be used. For example, human HR can be prepared by submitting bone marrow aspirations to "FICOLL HYPAQUE" centrifugation. Positive selection does not necessarily require the use of antibodies that recognize the specific determinants of CF. For example, B cells and monocytes may first be depleted of the CF-containing fraction after density gradient centrifugation, plastic adhesion, and washing of the FC receptor, then an antibody to the MHC-Class II antigen may be used to select positively the FC. The negative selection includes modifications to the protocol disclosed herein. For example, a cell preparation containing FC can be reacted with one or more antibodies directed to cell surface antigens not expressed by FC to the removal of non-FC. Antibodies can be used at a number of T cell, monocyte B cell and granulocyte markers. Examples of these antibodies include anti-CD4 and anti-TCR specific for T cells; anti-CD12, anti-CD19 and anti-CD20 specific for B cells; anti-CD14 specific for monocytes; and anti-CD16 and anti-CD56 specific for natural killer cells. These antibodies can be applied in any combination repeatedly or sequentially for the enrichment of the FC. During binding to the antibodies, the cells can be removed by adsorption to a solid surface coated with an anti-mouse antibody, since most of the monoclonal antibodies directed to the human cell surface markers are of mouse origin, or if the antibodies are conjugated with biotin, the cells bound to the antibody can be removed by a surface coated with avidin or streptavidin; or if the antibodies are conjugated in magnetic beads, the cells expressing the antigens recognized by the antibodies can be removed in a magnetic field (Harlow and Lane, 1988, Antibody: A Laboratory Manual, Cold Spring Harbor). Current methods for FC enrichment require a series of positive and negative selection steps, therefore, other sources of antigens associated with FC can be used. For example, brain tissue appears to contain the same anti-reactive antigens or antigens as that expressed by FC, and can be prepared for use as immunogens for the production of anti-human FC MAb. The brain tissue of any species can be obtained and prepared for use in immunization in the same way as described in Section 6.1.2, infra, except that the large tissue must be cut into small sections before homogenization. The second impediment is related to a deficient method for differential selection of the desired specific antibodies, that is, to select antibodies that are targeted to FC but much less to other blood cells. For the purpose of the present application, FCs are defined as cells derived from the bone marrow of approximately 8 to 10 microns in diameter, capable of improving the hemocytoblast graft and expressing Thy-1, CD3, CD8, CD45, CD45R, MHC Class II (low and intermediate levels), but lack other markers such as CD4, CD5, CD16, CD19, CD20, CD56, gammadelta-TCER and alpha-beta-TCR. MAb can be selected by binding assays where the antibodies bind to the FC but not to a lesser degree to other bone marrow cells including hemocytoblasts, T cells, B cells, macrophages, monocytes, granulocytes, red blood cells and platelets. The dyeing of the antibody can be determined by flow cytometry or any of the other detection methods known in the art. Alternatively, the antibodies can be selected because of their ability to deplete the function of the FCs as in an in vivo graft assay described in Section 6, infra. . 2 PRODUCTION OF ANTIBODIES Different methods can be used to produce polyclonal and monoclonal antibodies that recognize novel antigenic markers expressed by FC. Any method known in the art for the production of antibodies of these cells can be used. For the production of antibodies, different host animals can be immunized by injection with viable, purified or partially purified FC or brain tissue, cell preparations or fixed membranes, including, but not limited to those of rabbits, marmots, mice, rats , etc. Various adjuvants may be used to increase the immune response, depending on the host species, including but not limited to Freund mineral gels (complete or incomplete), such as aluminum hydroxide, surfactant substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacillus Calmette-Guerin) and Coryneacterium parvum. MAbs that are essentially homogeneous antibodies to the individual antigenic epitopes in the FCs can be prepared using any technique that provides means for the production of antibody molecules by continuous lines in the culture. These include, but are not limited to, the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256, 495-497), the most recent human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4: 72; Cote et al., 1983, Proc. Nati, Acad. Sci. USA 80: 2026-2030) and the EBV-hybridoma technique (Colé et al., 1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. , pages 77 to 96). Mabs can be differentially selected by selective binding to FC, but not to mature macrophages, granulocytes, monocytes, T cells, B cells, hemocytoblasts and dendritic cells, and / or by inhibition of FC activity. Antibody fragments that contain the binding site of the molecule can be generated by known techniques. For example, these fragments include, but are not limited to: F (ab ') 2 fragments that can be produced by pepsin diestion of the antibody molecule and F (ab') 2 fragments that can be generated by reducing the disulfide bridges of F (ab ') 2 fragments. A chimeric antibody is a molecule in which the different portions are derived from different animal species such as those having a variable region derived from a murine or rat MAb and a human immunoglobulin cosntant region. The techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984), Proc. Nati Acad.

USA 81: 6851; Nauberger et al., 1984, Nature, 312: 604-608; Takeda et al., 1985, Nature, 314-452-454) splicing the genes of a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can also be used. This approach is particularly useful if the antibodies are administered to humans. Chimeric antibodies exhibit fewer xenogeneic epitopes by inducing an anti-rodent Ig response when injected into man. Alternatively, the techniques described for the production of single chain antibodies (U.S. Patent Number 4,946,778; Bird, 1988, Science 242: 423-425; Huston et al., 1988; Proc. Nati. Acad. Sci. USA 85: 5879- 5883; and Ward et al., 1989, Nature 334: 544-546) can be adapted to produce single chain antibodies reactive to CF. Single chain antibodies are formed by linking the heavy and light chain fragment of the Fv region through an amino acid bridge, resulting in a single chain polypeptide. In addition, the whole antibody molecule or its Fab, (F (ab ') 2 Fragment Fv can be conjugated to any of a variety of compounds including, but not limited to, signal generating compounds such as fluorochrome, radioisotope, a chromophore, an enzyme, a chemiluminescent or bioluminescent molecule, etc. Alternatively, the whole antibody or its Fab, F (ab ') 2 ° Fv fragment can be conjugated to a cytokine that can improve or inhibit the biological activity of FC; or toxins of maenra that the FC expressing the corresponding antigens were selectively exterminated (Vitetta and Uhr, 1985, Annu Rev. Immunol., 3: 197). The methods that can be used to conjugate the irradiations, proteins, toxins, etc. in antibodies and antibody fragments are well known in the art (See, for example, U.S. Patent Nos. 4,220,450; 2,235,869; 3,935,074 and 3,996,345). . 3. USES OF MONOCLONAL ANTIBODIES TO HEMATOPOYETIC FACILITATORY CELLS A variety of uses of the MAbs are encompassed by the present invention. An antibody that exhibits exquisite specificity for CFs since it does not bind to T cells, B cells, NK cells, granulocytes, macrophages, monocytes, red blood cells, platelets and hemocytes, can be used to isolate CFs in a one-step affinity cell separation procedure. Antibodies or markers that are selectively expressed by FCs, that is, certain but not all blood cells also express it, and can be used effectively in combination with other methods such as density gradient centrifugation to significantly reduce delayed procedures and annoying that are currently used for the isolation of FC. For the practice of this aspect of the invention, a MAb can be conjugated to fluorochromes and used to screen for CF from a mixture of cells by flow cytometry using a fluorescence activated cell sorter or can be conjugated to biotin for use in separations of biotin-avidin or biotin-streptavidin. In the latter method, avidin or streptavidin is linked to a solid support such as an affinity column matrix or plastic surfaces. In addition, the antibodies can be coated with magnetic beads, reacted with a mixture of cells and the FCs bound to the antibody can be removed by a magnetic field. In addition, these MAbs can be conjugated to an enzyme for use in the immunohistochemistry. For example, certain disorders can be induced or sustained by an aberrant function of the FC, and the detection of the level of the FCs in the tissue sections can be of diagnostic value.

In addition, MAbs targeting FC markers can be used to isolate and identify the genes encoding these molecules. The antibodies can be used for selection expression libraries produced from the FCs for molecular cloning of the coding sequences (Seed and Aruffo, 1987, Proc Nati Acad Sci USA 84: 3365-3369). 6. EXAMPLE: GENERATION OF MONOCLONAL ANTIBODIES TO HEMATOPOYETIC FACILITATORY CELLS MURINE 6. 1. MATERIALS AND METHODS 6.1.1. ANIMALS The eight-week-old mice C57BL / 10SnJ (BIO), and B10.BR/SgSn (B10.Br) purchased from Jackson Laboratory (from Bar Harbor, Maine). Male four to eight week old Fischer 344 (F344) rats were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, Indiana). The animals were housed in a specific facility free of pathogens in the Biomedical Science Tower at the University of Pittsburgh. 6. 1.2. IMMUNIZATION AND FUSION OF CELLS Calvarium mouse brain tissue was obtained. The brain tissue was placed in 1 milliliter PBS for each 1 cubic centimeter of brain tissue, which was homogenized in a glass homogenizer. Then the brain emulsion was mixed with a Complete Freund's Adjuvant at a ratio of 1: 1 before injecting the animal. An 0.4 milliliter adjuvant of the homogenized mouse brain was emulsified. The injections to the rat were administered subcutaneously every 2 weeks for a total of four injections. Three days after the fourth injection, peripheral blood was tested to determine antibody production. The animals exhibiting the most intense activities were then selected for use for hybridoma fusion. The selected animals then administer an additional injection of a brain / PBS mixture subcutaneously in the absence of complete Freund's adjuvant. This injection was generally about 5 to 6 days after the fourth injection. Two days after this fifth injection, the spleens of the animals were harvested and fused with HGPRT myeloma cells (P3.653) using polyethylene glycol (Kohler and Milstein, 1975, Nature 256: 495). The cells were then distributed into microwell plates and grown in HAT medium (RPMI-1640 supplemented with 10 percent fetal bovine serum, 1 percent Pen / Strep, 1 percent L-glutamine, 1 percent of non-essential amino acids, 1 percent sodium pyruvate and HAT). The non-melted myeloma cells died due to their lack of HGPRT to use the salvage route. The non-fused spleen cells also died because they were unable to grow in vitro. The melted cells (hybridomas) grew in the microwells and their liquid culture supernatants were first tested to determine the production of rat antibodies. The culture supernatant liquid was selected to determine the presence of anti-mouse rat antibodies by incubating mouse bone marrow cells 10 ^ in small flow tubes with rat serum to block non-specific rat antibody staining. When 20 to 30 microliters of hybridoma culture supernatant fluid were added to each bone marrow tube, two separate tubes were tested for each supernatant fluid - one to select the production of IgG and the other for IgM production. After an incubation of 4 ° C for 45 minutes, the cells were washed twice at 1000 revolutions per minute for 10 minutes and the medium was decanted. Goat anti-rat IgG-FITC was added pre-culture, and anti-rat IgM-PE was added to the second tube. After 45 minutes of incubation at 4 ° C, the cells were washed twice and fixed in 0.4 milliliter of 1 percent paraformaldehyde for subsequent flow cytometric analysis. Controls included samples with single cells, IgG alone, IgM alone as evaluations of background dyeing and negative controls; and RAMB, Lyt2-FITC, and non-irradiated rat IgG and IgM MAbs against mouse antigens known as positive controls. Hybridomas were selected for the production of rat anti-mouse antibodies that were made counter-reactive with populations other than mouse bone marrow. Positive wells were further selected for antibodies directed to FCs in an in vivo assay. The selected hybridomas were cloned by limiting dilution. The cloned hybridomas were injected into nude mice primed with pristane for the production of ascites. 6. 1.3. PREPARATION OF MIXED POLYOGENIC CHEMISTRIES In order to classify and select the MAbs directed to the FCs, a preparation of the bone marrow cells of the donor was reacted with liquid hybridoma supernatants before their injection into the allogeneic mouse receptors. The allogeneic chimerism mixed in the receptors was used as an indicator of the presence of the MAb capable of exhausting the function of the FC. To prepare the mixed chimeras, the bone marrow from the long bones of the syngeneic mice (BIO) and the allogeneic mice (B10.BR) were harvested. The mice were euthanized with CO2 narcosis, prepared with 70 percent alcohol, and the long posterior bone (femoral and tibia) was removed. The marrow was detached from the bones using medium 199 (Gibco Laboratories Life Technology, Inc., of Grand Island, New York) supplemented with 50 microliters per milliliter of gentamicin using a 22 gauge needle. The mixture of medium (MEM) used to mechanically resuspend the bone marrow by gentle aspiration through an 18-gauge needle and suspension. filtered through a sterile nylon mesh gauze. The cells were then granulated at 1000 revolutions per minute for 10 minutes, resuspended in MEM, and counted. In normal allogeneic reconstruction, RAMB was used to deplete the B10 syngeneic bone marrow T cell (1:40 or appropriate dilution to 10 ^ cells per milliliter at 4 ° C temperature for 30 minutes). RAMB was prepared in the same manner as that described for the immunization of rats with mouse brains in Section 6.1.2, supra, with the exception that the mouse brain was used to immunize rabbits. Bone marrow allogeneic B10.BR cells were either untreated, depleted of RAMB, depleted of anti-Thyl.2 or with the treated hybridoma supernatant fluid. The bone marrow cells of the 10x10 * 5 donor were pelleted and the antibodies were added 1:10 in 1 milliliter. The media was preheated to 37 ° C so that incubation of the antibody was carried out at 37 ° C for 30 minutes. The cells were then washed in MEM, rotated at 1000 revolutions per minute for 10 minutes and resuspended in the guinea pig complement at 37 ° C for 30 minutes (Gibco Laboratories Life Technology, Inc., Grand Island, New York). The cells were washed twice, counted and resuspended in MEM at the appropriate concentration to allow the injection of 1 milliliter of total volume per animal. The syngeneic cells treated with RAMB were injected at 15x10 * ^ / animal while the allogeneic cells were administered at 15x10 * Vanimal within 4 to 6 hours after irradiation of the recipient animals at 9.5 Gy. Cell injections were through the lateral tail veins using a 27 gauge needle. 6. 1.4. CHARACTERIZATION OF CHIMERAS BY MEANS OF FLOW CYTOMETRY The recipients were characterized by grafts with syngeneic and allogeneic donor lymphoid elements using flow cytometry to determine the percentage of peripheral blood leukocytes (PBL) that carry MHC Class I surface markers (H-2b or H-2k). In short, the peripheral blood was collected in small vials of heparinized plastic serum. After vigorously mixing, the suspension was layered on 1.5 milliliters of room temperature lymphocyte separation (LSM) medium (Organon Technical, Kensigton, Maryland) and centrifuged at 20 ° C at 1700 revolutions per minute for 30 minutes. minutes The lymphocyte layer was aspirated from the saline-LSM interface and washed with the medium. Red blood cells underwent Used with ACK (used ammonium chloride / potassium carbonate stabilizer) and the remaining cells were stained with the appropriate MAbs for 30 minutes at 4 ° C and counterstained with a sandwich when required. Analyzes of the lymphoid cells of the spleen and thymus were carried out using a fluorescence activated cell sorter (FACS) (FACS II Becton Dickinson and Company, Mountain View, California). 6. 2 RESULTS The experiments described in the following sections used a mixed chimera model in which the recipient animals were irradiated lethally and transplanted with fixed doses of allogeneic donor cells and syngeneic donor cells. The percentage of the allogeneic chimerism, ie, the level of mixed chimerism was used as a reading of the activity of the FCs to activate the graft of the donor cell. It was previously reported that treatment with RAMB and complement of bone marrow preparation negatively affected its ability to be grafted into a recipient. Recently, a population of bone marrow cells has been identified which is referred to as FC that greatly improved the haematopoietic hemopoietic graft. In allogeneic bone marrow transplantation, hematopoietic hematopoietic cells purified with Sca-1 + alone were not able to perform the graft unless the FCs were co-administered. In addition, FCs did not possess hemocytoblast activity. Since RAMB appeared to deplete the FC and RAMB was an antiserum against the mouse brain, it was possible for mouse CFs to share certain common or counterreactive antigens with mouse brain tissues. Therefore, homogenized mouse brain tissues were obtained and used as immunogens in rats for the production of the counter-marker MAbs expressed by mouse FC. After several immunizations with the mouse brain tissues, the rats were sacrificed and their spleen cells were fused with HGPRT myeloma cells by polyethylene glycol. The resulting hybridoma cells were selected in the HAT medium and their liquid culture supernatants were tested for their ability to reduce the level of the donor bone marrow graft in allogeneic receptors as an indication of the presence of antibodies capable of eliminating FC. . The antibody selection procedure utilized a model of allogeneic mixed chimerism established where the recipient mice received TCD-syngeneic bone marrow cells plus allogeneic bone marrow cells treated with different antibodies. The level of allogeneic chimerism in the receptors was determined by the use of anti-MHC class I antibodies, and was used as an indication of the effects of the antibodies on the function of the FC. For example, an untreated preparation of the allogeneic donor bone marrow led to completely allogeneic allogeneic receptors, that is, mainly the H-2k allogeneic cells, with few syngeneic cells (H-2 ^) that were detected in the receptors (FIGURES 1A and IB). On the other hand, the allogeneic donor cells treated with RAMB (FIGURE 2A and 2B) or the anti-Thyl.2 antibody (FIGURES 3A and 3B) led to mixed allogeneic chimerism, indicating that these reagents depleted the FCs that were necessary to activate the allogeneic hematoclasto graft. In comparison with these controls, the bone marrow donor cells were treated with hybridoma supernatants and subsequently transplanted into allogeneic receptors to select the antibodies that would produce mixed alogeneic chimerism similar to the results obtained with the RAMB or anti-Thyl treatment. 2. Antibodies that did not reduce the level of complete allogeneic chimerism were discarded since they were not able to remove HR. Of the numerous hybridomas generated from those tested in the aforementioned assay, three hybridoma cell lines designated R7.6.2 (IgG2a), R340.3.1 (IgM) and R373.6.3 (IgM) were selected for further studies. These cell lines produced antibodies that were targeted to FC markers as evidenced by their ability to cause allogeneic chimerism mixed in recipients transplanted with donor cells treated therewith, while untreated donor cells produced completely allogeneic chimeras (Table I ). These results indicated that the three MAb were directed to the FC, capable of depleting the FC in the preparation of the donor cell and in turn causing a decrease in the capacity of the stem cells for the graft. Of a total of more than 150 classified hybridomas, only three clones produced antibodies that were preferentially ligated to the FCs.

TABLE I MONOCLONAL ANTIBODIES ADDRESSED TO FC CLONA # animal H-2b H-2 (syngeneic) (allogeneic) R7.6.2 # 956 18.90 79.12 # 961 12.12 86.98 # 962 12.48 91.18 # 964 10.66 93.26 # 966 27.35 70.34 # 967 12.58 88.18 R340.3.1 # 965 40.06 67.98 # 968 9.2 98.24 R373.6.3 # 773 85.54 13.91 # 774 97.28 0.54 # 775 35.89 57.57 # 974 99.76 0.38 Exhaustion # 272 50.78 48.02 of RAMB # 273 49.22 46.72 # 274 49.34 44.04 # 275 97.54 5.74 Exhaustion of # 969 63.34 33.56 Anti-Thyl.2 # 971 64.52 32.90 BIO Control 95.52 0.56 B10.BR Control 0.96 99.24 7. CELL LINE DEPOSIT The following hybridoma cell lines were deposited at American Type Culture Collection, Rockville, Maryland and assigned the following access numbers: Hybridoma Accession Number ATCC R7.6.2 HB11517 R340.3.1 HB11518 R373. 6.3 HB11507 The present invention should not be limited in scope by the exemplified embodiments that are intended as illustrations of the individual aspects of the invention. Of course, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. These modifications are intended to be within the scope of the appended claims. All publications cited herein are incorporated by reference in their entirety. 0 International Application No: PCT MICROORGANISMS Optional Sheet in relation to the microorganisms referred to on page 23, lines 1-20 of the description A. DEPOSIT IDENTIFICATION Additional deposits are identified on an additional sheet Name of the institution deposited: American Type Culture Collection Institution address deposited (including zip code and country) 12301 Parklawn Drive Rockville, MD 20852 United States Deposit date: December 10, 1993 Accession number: 11507 B. ADDITIONAL INDICATIONS (leave blank if not applicable). This information is continued on a separate sheet included C. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE: (if the indications are for all designated States) D. SEPARATE SUPPLY OF INDICATIONS: (Leave blank if it is not applicable) The indications mentioned below will be subject to International Department later (Specify the general nature of the indications, e.g., "Deposit Access Number") E. [x] This sheet was received with the International application when it was submitted (to be verified by the receiving office) (illegible signature) (Authorized Official) [] The date of receipt (of the applicant by the International Department) Authorized Official Form PCT / RO / 134 (January 1981) International Application No: PCT Form PCT / RO / 134 (continued) American Type Culture Collection 12301 Parklawn Drive Rockville, MD 20852 United States Access Number Deposit Date 11517 January 4, 1984 11518 January 4, 1984

Claims (7)

CLAIMS:

1. A monoclonal antibody, the antigen binding region from which competitively inhibits the immunospecific binding of the monoclonal antibody produced by the hybridoma R7.6.2 having an accession number of ATCC HB11517 to its reference epitope.

2. A monoclonal antibody, the antigen binding region from which competitively inhibits the immunospecific binding of the monoclonal antibody produced by hybridoma R340.3.1 having an accession number of ATCC HB11518 to its reference epitope.

3. . A monoclonal antibody, the antigen binding region from which competitively inhibits the immunospecific binding of the monoconal antibody produced by hybridoma R373.6.3 having an accession number of ATCC HB11507 to its reference epitope.

4. The monoclonal antibody produced by hybridoma R.7.6.2 as deposited with ATCC having an accession number HB11517.

5. The monoclonal antibody produced by hybridoma R340.3.1 as deposited with ATCC having an accession number HB11518.

6. The monoclonal antibody produced by the hybridoma R373.6.3 as deposited with ATCC having an accession number HB1150

7. 7. A method for generating monoclonal antibodies to antigens expressed by hematopoietic facilitator cells comprising immunizing an animal with homogenized brain tissue, melting the animal's spleen cells with a fusion partner cell line, and selecting the melted cells that secrete antibodies. capable of depleting facilitatory cells.