Human Antibodies - Volume 4, issue 1

Authors: Liu, Hua | Xu, Zuo-Liang | Wang, Yan | Yang, Li | Feng, Oi | Li, Yue | Wang, Ya-Ming | Zhang, Gui-Guo

Article Type: Research Article

Abstract: The production of anti-tumor human monoclonal antibodies (MAbs) by human-human or human-mouse hybridoma technology was studied. UC729-6, a human lymphoblastoid cell line, or NS-l, a mouse myeloma cell line, were fused with lymphocytes isolated from regional lymph nodes of 26 patients with breast or gastrointestinal cancer, resulting in 130 immunoglobulin-secreting human-human hybrids and 21 human-mouse hybrids. The supernatants of 88 hybrids were screened against a panel of cancer cells. The supernatants of 37 human-human hybrids and 2 human-mouse hybrids reacted with cancer cell lines. After three times subcloning, only one anti-breast cancer hybrid human MAb, IgG(λ) human-human hybridoma (MUBL-6), and …one anti–gastric cancer human MAb, IgM(λ) human-mouse hybridoma (HMG-1), were obtained. The antibody-secreting level was 1–4 μg/ml/24 h. Production of anti-breast cancer human MAbs by Epstein-Barr virus (EBV) hybridoma was also studied. Human lymphocytes were derived from draining lymph nodes of a breast cancer patient, whose serum antibody strongly reacted with tumor associated antigen (TAA). The enriched B cells were transformed with EBV in vitro. Positive antibody-secreting B cells were selected, expanded, and fused with heteromyeloma SHMD-33. The fusion frequency was 28/107 lymphocytes. Among them were 16 hybridomas secreting human immunoglobulin. After subcloning, 60% of the cloned hybridomas kept their antibody-secreting ability. Six observed hybridomas remained stable for more than 1 year in tissue cultures. The antibody-secreting level was 2.9–30 μg/ml/24 h. Supernatants from these hybridomas all reacted with breast cancer cell lines but not with gastric cancer cell lines. The supernatants from these hybridomas reacted with malignant cells in some breast cancer tissue sections but not with stroma or normal cells. In summary, the EBV hybridoma technique offers several advantages over other hybridoma systems for generating anti-tumor human MAbs. Show more

Keywords: human monoclonal antibody, human-human hybridoma, human-mouse hybridoma, EBV hybridoma, anti-tumor monoclonal antibody

DOI: 10.3233/HAB-1993-4101

Citation: Human Antibodies, vol. 4, no. 1, pp. 2-8, 1993

Authors: Kato, Masatoshi | Mochizuki, Katsumi | Hashizume, Shuichi | Tachibana, Hirofumi | Shirahata, Sanetaka | Murakami, Hiroki

Article Type: Research Article

Abstract: It has been known that the lung cancer–associated human monoclonal antibody HB4C5 comprises two lambda light chains of 30 and 32 kD and that the 30 kD species is exclusively responsible for the antibody activity. This study demonstrates that the two light chains were both N-glycosylated with glycosyl residues of different sizes, one of which was sensitive to neuraminidase and the other insensitive. Our unpublished data of DNA sequence for the light chain of this monoclonal antibody indicated that the light chain contains only one possible site for N-glycosylation, which located in the CDR-1. N-Deglycosylation of this monoclonal …antibody under the denaturing condition resulted in the complete conversion of the two light chains into one identical polypeptide chain of 26 kD. Activity of this monoclonal antibody was found to be significantly enhanced by N-deglycosylation. All the facts described above consistently indicate that the activity of this monoclonal antibody is interfered with by the attachment of bulky glycosyl residues at the antigen binding site on the light chain. The N-deglycosylated antibody was stable under the storage conditions employed, suggesting that the glycosyl residues attached to the light chain do not play any important biological role except for interference with the antibody activity of binding antigen. Show more

Keywords: monoclonal antibody HB4C5, light chain, N-glycosylation, antigen binding site, activation, heterogeneity

DOI: 10.3233/HAB-1993-4102

Citation: Human Antibodies, vol. 4, no. 1, pp. 9-14, 1993

Authors: Hagiwara, Hideaki | Yuasa, Hideo

Article Type: Research Article

Abstract: A novel cell line called Trioma, which can proliferate and secrete a human monoclonal antibody in the basal medium, was established. Trioma was generated by somatic cell hybridization with human × human hybridoma and A431c, which is a cell line able to grow autonomously in the basal medium. A Trioma called TriH8 has been kept growing in the DMEM/F-12 medium for over 1 year and stably producing stomach cancer–reactive human IgM into culture medium at 10 μg/ml. TriH8 has a characteristic cytological phenotype, that is, to diminish cell growth in the presence of epidermal growth factor.

Keywords: Trioma, human monoclonal antibody, human × human × human hybridoma, serum-free medium, tumor cell antigen

DOI: 10.3233/HAB-1993-4103

Citation: Human Antibodies, vol. 4, no. 1, pp. 15-19, 1993

Authors: Lund, John | Takahashi, Noriko | Hindley, Siobhan | Tyler, Ruth | Goodall, Margaret | Jefferis, Royston

Article Type: Research Article

Abstract: Oligosaccharide profiles for monoclonal chimeric mouse-human and mouse IgG2b antibodies produced in the mouse J558L cell line have been determined. These chimeric antibodies share the same mouse light chain and VH region and have a single, complex, oligosaccharide moiety attached to residue 297 of the heavy chain. Since the panel of chimeric antibodies included each of the human IgG subclasses and a set of IgG3 proteins having single residue differences, it was possible to evaluate the contributions of the protein template, the cell line, and the culture conditions to glycosylation. The study shows that the J558L cell line glycosylates …human heavy chains with oligosaccharides characteristic of mouse immunoglobulins. This includes a galactose alpha 1→73 galactose structure not found in human IgG and for which most humans have naturally occurring “anti-Gal” antibodies. The extent of galactosylation was very dependent on the culture conditions and was maximal for still culture. The findings have significance for the production of antibodies for in vivo administration. Show more

Keywords: chimeric IgG, glycosylation control

DOI: 10.3233/HAB-1993-4104

Citation: Human Antibodies, vol. 4, no. 1, pp. 20-25, 1993

Authors: Li, Yun | Ashby, Julian M. | Eremin, Oleg

Article Type: Research Article

Abstract: The selective cytotoxicity of the lysosomotropic leucine methyl ester and its lysosomal condensation product leucyl-leucine methyl ester have been used to investigate their effect on a range of lymphocyte subsets and on the cellular proliferation and secretion by immunoglobulin-secreting B cells from axillary regional draining lymph nodes of breast cancer patients. CD2+ , CD3+ , and CD8+ lymphocyte subsets were selectively reduced by the leucyl-leucine methyl ester treatment (CD2: 84.2–67.5%; CD3:76.I–62.3%; and CD8: 8.0–3.4%), but there was no significant reduction in the CD4+ and CD19+ subsets (CD4: 68.2–64.7%; and CD19: 22.6–33.2%). In the presence of mouse splenic …macrophages as antigen-presenting cells, rIL-2, IFN-gamma, and pokeweed mitogen-stimulated lymphocyte supernatant, leucyl-leucine methyl ester-treated lymphocytes showed a significant increase in 3H-thymidine incorporation and in the number of immunoglobulin-secreting B cells following coculture with the breast tumor cell line MCF-7. In this study, we have characterized some of the cellular and cytokine factors that are necessary for in vitro immunization of human B lymphocytes. Hopefully, this will enable human MAbs to be produced in vitro against tumor-associated antigens. Show more

Keywords: in vitro immunization, human monoclonal antibody, tumor-associated antigen, breast cancer

DOI: 10.3233/HAB-1993-4105

Citation: Human Antibodies, vol. 4, no. 1, pp. 26-30, 1993

Authors: Zhu, Yong | Jin, Boquan | Sun, Chen | Huang, Chuanshu | Liu, Xuesong

Article Type: Research Article

Abstract: Interleukin 6 (IL-6)/hybridoma growth factor (HGF) has been shown to be the requirement for growth of murine hybridomas in vivo or in vitro. In this paper, two kinds of conditioned media (CM) from the culture supernatants of a human fibroblast cell line CRL1506 and a cloned Epstein-Barr virus (EBV) transformed human lymphoblastoid cell line (LCL) N23 were found to have IL-6 activity by strongly promoting the growth, antibody secretion (increase of one- to three-fold), and cloning efficiencies of heterohybridomas secreting human monoclonal anti-hemorrhagic fever with renal syndrome virus antibodies and LCL. Since these CM contained no detectable IL-2, and IL-4 …had no effects on the growth of the cell lines, IL-6 was considered to be the main active component of the CM responsible for promoting hybridoma growth. This effect was further confirmed by IL-6–dependent cell line 7TD1 bioassay (IL-6 activity in the CM ranging from 1,000 to 10,000 units/ml). Moreover, we successfully established four EBV-transformed lymphoblastoid cell lines at single-cell level by adding an equal volume of CRL1506-CM to 10% FCS–RPMI1640 in limiting dilution. Finally, it is worth noting that the sensitivity of the heterohybridomas to the two kinds of CM was not the same and was not consistent with that of their parental myeloma cell lines. Thus, it suggests that the CM might contain more than one factor, and the choice of proper conditioned media should be very useful for human monoclonal antibody production. Show more

Keywords: conditioned media, hybridoma growth factor, heterohybridoma, cloning, antibody secretion

DOI: 10.3233/HAB-1993-4106

Citation: Human Antibodies, vol. 4, no. 1, pp. 31-35, 1993

Authors: Daifuku, Richard | Panacek, Edward A. | Haenftling, Katherine | Swenson, Wendy K. | Prescott, Amanda W. | Johnson, John L.

Article Type: Research Article

Abstract: MAB-T88 is a human monoclonal IgM antibody directed at the lipopolysaccharide of gram-negative bacteria. A protocol was designed to identify a group of septic patients with a very high likelihood of gram-negative bacteremia. All 6 patients entered in the protocol had a gram-negative source, and 4 of 6 had gram-negative bacteremia. In this patient population, MAB-T88 was shown to be safe with an effective half-life of 19.1 hours.

Keywords: gram-negative, IgM, lipopolysaccharide, monoclonal antibody, sepsis

DOI: 10.3233/HAB-1993-4107

Citation: Human Antibodies, vol. 4, no. 1, pp. 36-39, 1993