Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/34—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood group antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• Hemolytic disease erythroblastosis fetalis
• HDN new born infants
• 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.
• 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.
• the population of. such naturally- sensitized serum donors is rapidly decreasing.
• 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.
• 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.
• these antibodies are also useful for routine blood group typing in the blood bank.
• 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 e.g., 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.
• 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
• ⁇ ?NAT10 generate immortalized lymphoblastoid cells, which can be used in the subsequent fusion with a fusion partner.
• 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, S ⁇ hlo et al. (1980), PNAS USA 77:6841-6845; and Cro ⁇ e 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 ⁇ ytotoxic 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.
• PBS phosphate buffered saline
• 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,
• 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.
• SRBC sheep red blood cells
• 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 Is ⁇ ove'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) .
• ELISA enzyme-linked immunosorbent assay
• Rh(D) antigen-specific B-lymphoblastoid cells were selected by rosetting with papain-treated O HRBC.
• 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
• 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 sub ⁇ loned 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.
• 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.
• a hybrido a designated ElO-l was deposi t ed at the ATCC on November 22, 1983 and granted Accession No. HB-8435.

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• 1984
  • 1984-11-26 EP EP19850900333 patent/EP0162918A1/de not_active Withdrawn
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