THE PRODUCTION OF HUMAN MONOCLONAL ANTIBODIES

Abstract

The general procedures for production of human monoclonal antibodies by immortalization of B lymphocytes using EBV transformation or cell fusion were established. In the first method, B cells were enriched prior to EBV transformation by T cell depletion, involving incubation of peripheral blood lymphocytes with OKT 3 and complement. Plating of EBY-infected cells at a low density of 5 x 105 cells/ml in culture medium supplemented with 10 % FCS allowed for efficient transformation of B cells. Antibodies against blood group 'A' erythrocytes, Hep G2 hepatocellular carcinoma and CEM acute lymphoblastic leukemia cell line were obtained from EBV-transformed normal PBL, without deliberate immunization. An EBV-transformed clone obtained after in vitro stimulation in mixed lymphocyte culture, produced cytotoxic antibodies against lymphoblastoid cells of A2/B 13 HLA phenotype. Three clones producing antibodies against hepatitis B surface antigen were obtained by EBY transformation of in vivo presensitized lymphocytes. For the second method of cell fusion, the mouse myeloma NS-1 was selected as the fusion partner as it generated the largest number of hybrids when fused with PBL. Fusion using 47 % Merck PEG 4000 and 7.5 % DMSO with a short time exposure of 90 seconds was suitable. Moreover, higher hybrid yield was obtained for cells plated at low density of 1 x 105 cells/well, with mouse macrophages as feeder cells. Fusion frequency of 10-5 was obtained under the conditions optimized. A heteromyeloma generated from fusion of PBL stimulated in MLC, produced cytotoxic antibodies against lymphoblastoid cells of A33/B 17 HLA phenotype. In vitro immunization conditions were investigated to obtain and expand specific antibody-producing B lymphocytes. Culture medium, supplemented with 25 % phytohemagglutinin-stimulated lymphocyte culture supernatant, 10 % monocyte-conditioned medium, 100 units/ml y-interferon and 1:500 diluted pokeweed mitogen, supported in vitro sensitization and stimulation of B cells. OKT 8-depleted PBL was immunized in vitro with HBsAg and three clones producing anti-HBs antibodies were obtained after EBV transformation. One of the clones ( ?s/24) was subcloned twice and studied. Its culture supernatant was found to have anti-HBs activity of 97 mIU/ml and produced 0.6 µg/ml IgM per 106 cells per day. It was directed against the common 'a' determinant of HBsAg. Cell growth was maintained by combined fusion with NS-1, but antibody production was not sustained. Twelve heteromyelomas, designated as CY heteromyelomas, were generated from fusions of PBL with NS-1 and had stopped antibody secretion. The effect of 5-azacytidine on the hybridomas was tested and treatment with 2 µM 5-azacytidine reactivated 2 CY clones to produce human antibodies. B cell clones obtained were also used to study growth factor production. Supplements of supernatants from clones of B lymphoblastoid cell line, CSH BLCL, and CY heteromyelomas affected the proliferation of PBL in vitro. Four CSH clones and one CY clone were stimulatory, while three other CSH clones were found to be inhibitory. The heteromyelomas also served as useful target cells in the study of B cell surface antigens. A panel of monoclonal antibodies was obtained from the organizers of the Fourth International Conference on Human Leucocyte Differentiation Antigens (I 989). The CY heteromyelomas reacted with 7 % of the panel of 129 antibodies tested. Reactivity pattern with the antibodies was different for the 12 CY clones. Hence, specific clones secreting human monoclonal antibodies could be established by EBV transformation and/or fusion, using PBL that has been in vivo presensitized or immunized in vitro. Moreover, other clones produced can be applied to several areas of research, such as production of growth factors and study of B cell surface antigens, which may be beneficial to the understanding of B cell immune response.