HUMAN MONOCLONAL ANTIBODY AGAINST Rh(D) ANTIGEN AND ITS USES

Abstract: Human monoclonal antibodies specific for the Rh(D) antigen. These antibodies are particularly useful for preventing isoimmunization of Rh<-> mothers who have given birth to Rh<+> children.

Description: HUMAN MONOCLONAL ANTIBODY AGAINST Rh(D) ANTIGEN AND ITS USES Background of the Invention 1. Field of the Invention Hemolytic disease (erythroblastosis fetalis) in new born infants (HDN) can occur when the mother is Rh and the child is Rh . The fetal red blood cells, which carry the Rh antigen, can enter maternal circulation at time of delivery, abortion or intramutation, for example amniocentisis and can result in isoimmunization of the mother. The resulting maternal antibodies can then enter the fetal circulation during subsequent pregnanc(ies) and react with the fetal red blood cells, causing massive hemolysis and symptoms of HDN. An effective treatment has been developed for reducing the incidence of HDN. At each delivery from a + — high-risk parental combination (Rh father, Rh mother) , the mother is injected with anti-Rh antibodies obtained from the immunoglobulin fraction of immunized serum donors, e.g., sensitized Rh "" mothers who have recently given birth to Rh children. Injection of the anti-Rh antibodies causes rapid clearance of the fetal Rh cells from the maternal blood and minimizes the immunogenic priming of the mother. Heretofore, the antibodies administered to Rh " mothers have been derived primarily from the serum of sensitized Rh " women who had recently given birth to Rh children. As a result of the success of this treatment, however, the population of. such naturally- sensitized serum donors is rapidly decreasing. While suitable antisera may be derived by hyperimmuni- zation of Rh donors, the use of human volunteers is clearly undesirable, an d new sources of human anti-Rh antibodies are urgently required. 2. Description of the Prior Art The use of anti-Rh antisera derived from naturally-sensitized and hyperim unized donors for passive immunization has been reported. See, e.g., Clarke et al- (1963) Brit. Med. J. 1:979-984; Freda et al. (1964) Transfusion 4:26-32; oodrow et al. (1965) Brit. Med. J. 1:279-283; and Pollack et al. (1968) Transfusion 8:151-153. U.S. Patent application Serial No. 457,795 filed January 13, 1983, describes the preparation of heteromyeloma cells and their use in producing human-human hybridomas. A particular heteromyeloma cell designated SHM-33 was deposited at the ATCC on September 13, 1983 and granted accession no« CRL-1668 ± n connection with said patent application. Summary of the Invention Human monoclonal antibodies specific for the human Rh(D) antigen are provided. The antibodies are produced from a hybridoma resulting from the hybridization of an immortalized cell line and a human B-lymphocyte sensitized to Rh(D) antigen. Such antibodies are particularly useful for preventing isoimmunization of an Rh patient to Rh(D) antigen. The anti-Rh antibodies are administered in an amount sufficient to inhibit the immunogenic effect of the Rh(D) antigen. In addition, these antibodies are also useful for routine blood group typing in the blood bank. OMPI Description of the Specific Embodiments Methods are provided for the production and use of human monoclonal antibodies specific for the human Rh(D) antigen. The D antigen is the predominant Rh antigen, although a number of others also exist. Persons who are D~ " are commonly referred to as being Rh " , although they will usually possess at least some of the other antigenic determinants of the Rh locus. The subject monoclonal antibodies are obtained by fusion of human B-lymphocytes from an Rh{D)~ donor sensitized with the Rh(D) antigen and an appropriate fusion partner. Conveniently, the sensitized B-lymphocytes are obtained from an Rh "" patient who has recently given birth to an Rh child. Preferably, the mother will have previously given birth to one or more additional Rh children, and will have a significantly elevated anti-Rh(D) serum titer. The peak serum titer will occur from one week to three months after delivery. Alternatively, the sensitized B-lymphocytes may be obtained from any Rh(D)~ human host immunized with the Rh(D) antigen. Conveniently, the host will be immunized subcutaneously at one or more sites with injections of about 0.5 to 10 ml, usually about 5 ml, of whole Rh(D) blood. One or more booster vaccina¬ tions may be given at intervals ranging from 48 hours to three weeks. Specific methods for immunization are taught in Woodrow et al. (1965) , supra. Blood is obtained from the donor by venipuncture techniques, and peripheral blood lymphocytes are separated on a Picoll-Hypaque gradient. The T-lymphocytes may then be removed by E-rosetting with sheep red blood cells, yielding an enriched B-lymphocyte population. The B-lymphocytes can be transformed with Epstein-Barr virus (EBV) to OMPI ^?NAT10 generate immortalized lymphoblastoid cells, which can be used in the subsequent fusion with a fusion partner. A wide variety of fusion partners may be employed which provide for the secretion of human immunoglobulins. Desirably, the immunoglobulin which is secreted will be IgG, although IgM or IgA may also find use. The production of IgD and IgE is uncommon and not clinically useful. The fusion partner may be a mouse myeloma line, a heteromyeloma line (see Application Serial No. 457,795), or a human myeloma or other immortalized line, as described in PCT Application No. 81/00957, Sσhlo et al. (1980), PNAS USA 77:6841-6845; and Croσe et al. (1980), Nature 288:488-489. Desirable characteristics of a fusion partner are high efficiency of fusion to provide for a high proportion of immunoglobulin-producing hybridomas, absence of the production of individual chains or immunoglobulins unassociated with the immunogiobulin of interest, and the maintenance of the capability of continuously secreting the desired immunoglobulin over long periods of time. Illustrative fusion partners include the mouse myeloma cell lines X63-Ag8.653, P3-NSl/lAg4 and S194/5.XX0.BU1; and human fusion partners, such as UC729-6 and SKO-007. Particularly preferred are mouse-human heteromyeloma lines, such as SHM-D33. The fusion is carried out in the presence of polyethylene glycol for a short period of time. The polyethylene glycol is then removed, and the cells are subjected to selective conditions which are σytotoxic to the parent cells, but not to fused hybrid cells, e.g. HAT, HAT and ouabain, etc. Hybrid cells which grow out from the selective media are seeded in individual wells and their supernatants screened by any convenient technique for the monoclonal antibodies of interest. Cells secreting monoclonal antibodies specific for Rh(D) antigen are then cloned by limiting dilution procedures, and the clones producing the highest level of specific antibody expanded. The antibodies may then be further characterized as to classes, subclasses and type. The antibodies may be purified by any convenient technique, such as chromatography, electrophoresis, precipitation and extraction, or the like. The antibodies may be employed without further change after purification. The hybridomas may find use other than for production of the immunoglobulin. The hybridomas may be used as a source for the DNA or mRNA encoding the rearranged, activated anti-Rh(D) immunoglobulin genes, which may be isolated, cloned by recombinant DNA techniques and transferred to other cells for the production of the specific immunoglobulin. The anti-Rh(D) antibodies of the present invention may find use in screening blood samples for the presence of the Rh(D) antigen or antibody employing any one or a wide variety of conventional immunoassays. More importantly, the IgG antibodies of the present invention will be employed to inhibit immunogenic priming of an Rh~ mother about to give birth to an Rh child. After each delivery, the mother is injected with the anti-Rh(D) monoclonal antibodies, causing rapid clearance of the Rh(D) fetal erythrocytes from the maternal blood. The injections should be given within 72 hours of delivery to effectively inhibit primary immunization and prevent the subsequent development of anti-Rh(D) antibodies in the maternal circulation. The dosages needed is variable depending on the number of fetal cells actually entering maternal circulation at the time of delivery. It is estimated to be 100 - 300yg per treatment. The antibody may be formulated in any physiologically acceptable medium. typically phosphate buffered saline (PBS) , serum or water. The following examples are offered by way of illustration and not by way of limitation. EXPERIMENTAL B-lymphocytes were obtained from a 27 year old female patient having type A, Rh~ blood (genotype: cde/cde or rr) two weeks after delivery in her fifth pregnancy. The baby's blood type was O, Rh . The patient's anti-Rh(D) serum titer was 1:512 three weeks prior to delivery and 1:1024 two weeks after delivery. The B-lymphocytes were isolated as follows. Twenty cc of the patient's peripheral blood was obtained by routine venous puncture and collected in heparinized tubes without preservative. The peripheral blood mononuclear cells were separated from red blood cells and granulocytes on a Picoll-Hypaque gradient, 7 yielding 2 x 10 lymphocytes. The T-lymphocytes were then removed by mass-rosetting with neuraminidase- treated sheep red blood cells (SRBC) , yielding 2 x 10 • cells highly enriched for B-lymphocytes. The enriched B-lymphocytes were then transformed with Epstein-Barr virus (EBV) in supernatant culture fluid from the B95-8 marmoset cell line, added at 20% final concentration in Isσove's medium containing 20% fetal calf serum (FCS) . After 3 weeks, a polyclonal, permanently established B-lymphoblastoid cell line was obtained, the supernatant culture fluid of which was positive by enzyme-linked immunosorbent assay (ELISA) for reactivity with type O human red blood cells (HRBC) . Rh(D) antigen-specific B-lymphoblastoid cells were selected by rosetting with papain-treated O HRBC. These cells were then re-incubated and allowed to increase in number. After growth for an additional 22 days, 1 x 7 6 10 B-lymphoblastoid cells were fused with 5 x 10 SHM-?D33 human-mouse heteromyeloma cells (see Application Serial No. 457,795) in the presence of polyethylene glycol, as described by Oi and Herzenberg (1979) In: Selected Methods in Cellular Immunology (B. B. Mishell and S. M. Shiigi, eds.) W. J. Freeman & Co., San Francisco, Calif, pp. 351-372, with the following modifications. The cells were each washed twice with 50 ml calcium- and magnesium-free PBS (PBS-CMF) (Schneiderman et al. (1979) , Somatic Cell Genetics 5:263-269). The cells were then mixed, washed with 10 ml PBS-CMF, and 1 ml of 40% (w/v) polyethylene glycol 1540 in PBS-CMF was added to the pellet with gentle stirring for 1 minute. After one more minute, 2 ml of PBS-Iscove's medium (without fetal calf serum) were added at 1 ml/rain, and 8 ml of PBS over the next 2 minutes. The final pellet was resuspended in selection medium and added to 96-well plates at a concentration of 2 x 10 cells/well. Mouse thymocytes were used as 5 feeders at 10 /well. Selection was carried out in HAT medium (Littlefield (1964) Science 145:709-710) and 5 —7 x 10 M ouabain. After 15 days, viable hybrids were seen growing in 80% of the wells, and 100% of these were producing human mu and/or gamma heavy chains when assayed by ELISA. After 22 days, growth was evident in 98% of wells and 100% of these were human Ig producers. After one month, 100% of wells showed viable growth with 100% Ig producers. Of 60 wells tested, 60 (100%) were producing gamma heavy chain and 45 (75%) were producing mu heavy chain; 90% of these supernatant fluids gave positive hemagglutination tests with papain-treated 0 HRBC, and hemagglutination tests carried out on a panel of HRBC of known blood type antigenicity confirmed the specificity of these human antibodies for Rh(D) antigen. OMPI A total of 12 specific antibody-producing hybrid cultures were subcloned at 5 cells/well in two microtiter plates each. After from 2 to 3 weeks the first assays were carried out on these subcloned plates, revealing that 6 of 12 subσloned plates were producing only gamma heavy chain, whereas the other 6 were producing both mu and gamma heavy chain. Wells in all 12 subcloned plates continued to produce specific anti-Rh(D) antibody, as determined by positive hemag- glutination tests with 0 HRBC. According to the present invention, human monoclonal antibodies are provided which are specific for the Rh(D) antigen on human red blood cells. The hybridomas producing these antibodies may be stably maintained over long periods, providing a constant source of antibodies having well defined character¬ istics. Since the monoclonal antibodies are human, they may be used to prevent isoimmunization of Rh " mothers giving birth to Rh children without causing a significant immune response or shock to the human host. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims. A hybrido a designated ElO-l was deposi t ed at the ATCC on November 22, 1983 and granted Accession No. HB-8435.

Claims: WHAT IS CLAIMED IS: 1. Human monoclonal antibodies specific for / Rh(D) antigen. 2. Antibodies as in claim 1 which are IgG. 3. A hybridoma capable of producing 5 antibodies specific for Rh(D) antigen on human red blood cells, said hybridoma produced by hybridization of an immortalized cell line and a human B-lymphocyte sensitized to Rh(D) antigen. 4. A hybridoma as in claim 3 wherein said 10 immortalized cell line is a mouse-human heteromyeloma. 5. A hybridoma as in claim 4, wherein the B-lymphocyte is transformed by EBV prior to fusion. 6. A method for preventing isoimmunization of an Rh~ patient to Rh(D) antigen, said method 15 characterized by administering human monoclonal antibodies specific for Rh(D) antigen to said patient in an amount sufficient to inhibit the immunogenic effect of the Rh(D) antigen.

Inventors: BRON DOMINIQUE G, KAPLAN HENRY S, TENG NELSON H, BRON, DOMINIQUE, G., KAPLAN, HENRY, S., TENG, NELSON, H.
Applicants: UNIV LELAND STANFORD JUNIOR, THE BOARD OF TRUSTEES OF THE LELAND STANFORD JR. U
Application year: 1984
Main classification: C12P21
IPC codes: A61K38, C07K16