The Construction and Use of a Human-Mouse Myeloma Analogue Suitable for the Routine Production of Hybridomas Secreting Human Monoclonal Antibodies

Abstract

A human-mouse myeloma analogue termed HMMA2.11TG/O was constructed by fusion of the mouse myeloma cell line P3x63Ag8.653, a mutant derivative of MOPC21, with bone marrow mononuclear cells from a patients with IgA myeloma. The HMMA2.11TG/O cell line is resistant to 6-thioguanine and ouabain and sensitive to HAT. The cell line secretes no detectable immunoglobulin and has a hybrid karyotype and cell surface phenotype. An average fusion efficiency for growth of hybridomas of 1/17,000 fused cells was obtained in fusions with human peripheral blood mononuclear cells (PBM), Pokeweed Mitogen (PWM) stimulated PBM, and Epstein-Barr Virus (EBV) transformed polyclonal B cell lines. Over 75% of hybrids secrete detectable immunoglobulin and the cloning efficiency of the hybrids at 1 cell/well averages 25%. Antibody secreting cloned hybridoma cell lines were obtained by fusion directly with PBM from an immunized volunteer and by fusion with in vitro, secondarily immunized, EBV transformed polyclonal cell lines. Five hybridomas secreting human monoclonal IgM anti-tetanus antibodies and 2 secreting human monoclonal IgG anti-tetanus antibodies were selected and cloned from 6 fusions performed specifically for anti-tetanus antibody. Immunoglobulin and antibody secretion by cloned hybrids has been stable for 5-10 months at present. Immunoglobulin and antibody secretion in routine cultures passaged every 3-4 days has been 8-42 micrograms/ml. This human-mouse myeloma analogue should prove useful for the routine production of human monoclonal antibodies.

Truncation of the Cytoplasmic Domain Induces Exposure of Conserved Regions in the Ectodomain of Human Immunodeficiency Virus Type 1 Envelope Protein

“…Since all of the constructs with the FS exhibited increased binding of 17b and 48d even in the absence of CD4, we tested MAbs directed against other regions of Env to further characterize the conformational changes in the ectodomain resulting from the introduction of the FS mutation into the cytoplasmic domain of gp41. Figure 6 shows that the binding of conformation-dependent MAb F105, whose epitope overlaps the CD4-binding site in gp120 (7,8,(41)(42)(43), was enhanced by the FS mutation in all of the X4 (Fig. 6A), R5 (Fig.…”

Human Monoclonal Antibodies toPseudomonas aeruginosaAlginate That Protect against Infection by Both Mucoid and Nonmucoid Strains

“…Cultures that continued to test positive were then fused with a humanmouse myeloma cell line, HMMA 2.5, to generate hybridomas as previously described (24). After fusion, cells were cultured in microwell plates with growth medium (RPMI 1640 supplemented with 20% FBS and hypoxanthine-aminopterin-thymidine and oubain) for selection of fused cells.…”

Human hybridoma technology

“…Two of these cell lines, SHM-D33 27 and HMMA 2.5, 28 are frequently used for production of human mAbs. These cell lines are a nonsecreting mouse × human heteromyeloma that is resistant to ouabain and produces stable hybridomas upon fusion with human EBV-transformed B cells.…”