Human Antibodies - Volume 4, issue 2

Authors: Tachibana, Hirofumi | Shirahata, Sanetaka | Murakami, Hiroki

Article Type: Research Article

Abstract: Hybrid antibodies, including bifunctional antibody, were obtained by fusing two different drug-resistant clones of human hybridomas HB4C5 and SU-1. One hybridoma, producing human IgM-class monoclonal antibody reactive to porcine carboxypeptidase A (Cpase), was a 6-thioguanine resistant clone (6T-C5) of HB4C5 cell line. Another, secreting human IgM-class anti–double-stranded DNA (ds DNA) monoclonal antibody, was a 5-bromodeoxyuridine resistant clone (5B-SU) of SU-1. Hybrid hybridomas were generated by fusing the 6T-C5 and 5B-SU lines and screened by HAT selection. Among many hybrid hybridomas, A9C11 cells produced bifunctional antibody having dual specificity for Cpase and ds DNA, and the light chains of the antibody …consisted of the only ones derived from 5B-SU antibody, indicating that bifunctional antibody could be generated by heterologous association of heavy and light chains. Show more

Keywords: light chain, heterologous association, human monoclonal antibody, bifunctional antibody

DOI: 10.3233/HAB-1993-4201

Citation: Human Antibodies, vol. 4, no. 2, pp. 42-46, 1993

Authors: Brams, Peter | Royston, Ivor | Boernert, Paula

Article Type: Research Article

Abstract: Induction of antigen-dependent IgM and IgG responses by human in vitro spleen cell cultures were supported by addition of defined lymphokines. Whereas exposure to IL-2 alone throughout both immunization and cultivation period supported the highest antigen-directed responses, antibodies of higher relative affinity to the immunizing antigen were secreted under conditions of shorter IL-2 exposure. Continuous presence of IL-2 supported the antigen-driven responses for up to 15 days, the later part of which was characterized by a very low relative affinity index value. IL-2 supported cultures produced up to 55 times and 36 times more IgM and IgG, respectively, than cultures …without added IL-2. Addition of IL-2-neutralizing antibodies throughout the cultivation period abrogated all responses. Addition of IL-4 alone had negligible effect on the antigen-directed IgM responses but supported antigen-independent IgG responses. Neutralization of IL-4 alone had negligible effect on the IgM and IgG responses, whereas neutralization of IL-4 during IL-2 support, resulted in reduction of antigen dependency of responses. Delayed IL-4 neutralization (24 hours) restored some of the antigen sensitivity. IL-4 reduced IL-2-induced, antigen-directed immunoglobulin responses but supported increased antigen-induced, antigen-directed responses, resulting in maximal antigen-specific responses. IL-4 reduced the IL-2-induced immunoglobulin production. IL-2 supported cell division, whereas IL-4 supported prolonged survival. Flow cytometry tests showed that IL-2 primarily induced CD8+ cells (Tsuppressor/cytotoxic ) and OKT-10+ cells (plasma cells) cells, whereas the number of B1+ cells (B cells) decreased. IL-4 induced slight increases in the amount of B1+ cells and CD4+ cells (Thelper ). Show more

Keywords: In vitro immunization, human lymphocytes, lymphokine requirements, IL-2, IL-4

DOI: 10.3233/HAB-1993-4202

Citation: Human Antibodies, vol. 4, no. 2, pp. 47-56, 1993

Authors: Brams, Peter | Royston, Ivor | Boerner, Paula

Article Type: Research Article

Abstract: A protocol by which a human spleen cell culture could be subjected to both a primary and a secondary type immunization in vitro has been developed. Primary immunized cultures required the addition of autologous fresh cells at the time of antigen re-exposure for generation of optimal antigen-specific IgG responses. Antigen re-exposure (secondary stimulation) was done 10 days after initial immunization. Subsequent responses were characterized by novel or greatly enhanced antigen-directed IgG responses compared to primary antigen stimulation. Re-exposure of antigen to 10-day old human spleen cell cultures, without addition of fresh cells, did not result in measurable responses. Lethal irradiation …of the fresh cell component of a restimulation culture showed that the IgG responses were derived from the original culture and not the fresh cells. The presence of antigen during both the primary and the secondary immunization was a prerequisite for induction of antigen-directed IgG responses, as spleen cell preparations that had been cultivated without antigen, during the primary stimulation period or the secondary stimulation period or both, did not show antigen-dependent IgG responses after the secondary cultivation. Induction of IgG responses by restimulation was supported by IL-2, but only when IL-2 was added to both the primary and the secondary culture according to a strict protocol. IL-4 added during the second antigen exposure supported antigen-directed responses, whereas IL-4 added during the initial antigen exposure abrogated all antigen-dependent responses. Show more

Keywords: In vitro priming, human spleen cells, IgG responses, restimulation

DOI: 10.3233/HAB-1993-4203

Citation: Human Antibodies, vol. 4, no. 2, pp. 57-65, 1993

Authors: Mevissen, Marcel L.C.M. | Kwekkeboom, Jaap | Goormachtig, Elly | Lindhout, Ernst | de Groot, Cornelis

Article Type: Research Article

Abstract: Exposure of human B cells to Epstein-Barr virus (EBV) usually results in low frequencies of transformed cells. The transformed cells can be cloned poorly by limiting dilution, even when feeder cells are used. In recent years it has become clear that growth and antibody production of EBV-transformed cells are influenced by auto- and paracrine growth factors. Therefore, supernatants from the lymphoblastoid B cell lines JY and Raji were used as a source of growth factors to investigate their effect during EBV transformation of human B cells and consequently on cloning by limiting dilution of these transformed cells. Initial experiments to …clone three established EBV-transformed B cell lines showed a strong increase in outgrowth of the number of cells in the presence of the supernatant (range: 1 per 2–8 of the originally plated cells) as compared to cells cultured without the supernatant (range: 1 per 17–100 of the originally plated cells). Transformation efficiencies of freshly isolated tonsil B cells were not influenced by the supernatant and were generally less than 1%. In contrast, transformation efficiency was increased up to 9.4% if B cells were both transformed and cultured in the presence of the supernatant. Cloning efficiencies increased if the cells used were transformed in the presence of the supernatant. Best results were seen when the supernatant was present during transformation and cloning of the cells. The presence of the supernatant during transformation and/or cloning of the B cells dramatically enhanced the number of B cells secreting IgG. Cloning of two established tonsil B cell lines resulted in a large number of B cell clones. Nineteen randomly chosen B cell clones were tested for their immunoglobulin and cytokine production patterns. It was found that neither one of these tested clones showed the same production pattern. IL-4 and IL-5 producing B cell clones were found. The secretion of IgG was correlated with the capacity to produce IL-1 (8 out of 10 clones), while IgM secretion was correlated with a lack of IL-1 production (4 out of 4 clones). These results indicate that supernatants derived from continuously growing B cell lines can be used to obtain large numbers of IgG-secreting transformed B cells. The use of these supernatants probably does not selectively favor the growth of a specific subpopulation of B cells. Show more

Keywords: Epstein-Barr virus transformation, human B cells, antibody, cytokines

DOI: 10.3233/HAB-1993-4204

Citation: Human Antibodies, vol. 4, no. 2, pp. 66-73, 1993

Authors: Bender, Eckhard | Woof, Jennifer M. | Atkin, Julie D. | Barker, Mike D. | Bebbington, Chris R. | Burton, Dennis R.

Article Type: Research Article

Abstract: The combinatorial phage library approach to immunoglobulin repertoire cloning recently made it possible to isolate gene fragments encoding human immunoglobulin G1 Fabs binding with high affinity to specific antigens. Here we describe the construction of genes encoding whole human anti–tetanus toxoid antibodies based on one of these gene fragments and the efficient expression of these constructs by co-transfection of separate heavy and light chain vectors into a Chinese hamster ovary cell line constitutively expressing a viral transactivator protein. This system will be generally useful for the rapid analysis of recombinant antibodies derived from combinatorial libraries.

Keywords: human antibody, immunoglobulin repertoire cloning, CHO cells, tetanus toxoid

DOI: 10.3233/HAB-1993-4205

Citation: Human Antibodies, vol. 4, no. 2, pp. 74-79, 1993

Authors: Ferraro, Alicia S. | Newkirk, Marianna M.

Article Type: Research Article

Abstract: Peripheral blood B cells from normal individuals have been less than ideal as a resource for “fusible” cells for the generation of human hybridoma antibodies. In vitro stimulation of normal peripheral blood B cells by CD4+ T cells (HUT 78) that had been activated by a solid phase anti-CD3 monoclonal antibody (OKT3) was investigated to see if differentiation of B cells would result in an increased pool of B cells that could be immortalized. A comparison of the rate of successful fusion, an estimation of the frequency of the fusible cells from the input peripheral blood, and the amount of …immunoglobulin secreted both in vitro, prior to fusion, and by the resulting clones in two different in vitro immunization protocols, with and without activated T cells indicated that inclusion of the activated T cells provided the necessary help to drive the B cells to a fusible state. Stimulation by activated T cells improves the efficiency of generating B cell hybrid clones from peripheral blood 10-fold compared to in vitro immunization with antigen alone. Show more

Keywords: human monoclonal antibodies, anti-CD3 activated T cells

DOI: 10.3233/HAB-1993-4206

Citation: Human Antibodies, vol. 4, no. 2, pp. 80-85, 1993

Authors: Ditzel, Henrik | Erb, Karin | Leslie, Graham | Jensenius, Jens Chr.

Article Type: Research Article

Abstract: The large size of human IgM monoclonal antibodies (MAbs) may impede the tumor-localizing capacity. A procedure is described for the preparation of antigen-binding monomeric (IgMm ) and half-monomeric (IgMI½m fragments from two human IgM MAbs, COU-1 and D4213. The fragments retained binding activity against colon carcinoma. Six different reducing reagents (dithiotreitol, 2-mercaptoethanol, 2-mercaptoethylamine, L-cysteine, metabisulphite, ascorbic acid) were investigated over a range of concentrations, pHs, and incubation periods. The reduced IgM preparations were alkylated with iodoacetamide and fractionated by high-performance gel permeation chromatography. The fractions were directly collected on ELISA plates coated with extracts of colon cancer cells. Antigen-binding …IgMm and IgMI½m fragments were obtained after treatment with mercaptoethanol, mercaptoethylamine, metabisulphite, and cysteine. IgMm and IgMI½m fragments were also obtained after dithiotreitol treatment. These fragments were, however, nonreactive. The pH during the reduction was important for optimal yields of the fragments. The fragments obtained with 2-mercaptoethanol and mercaptoethylamine were most effective in binding to the cancer cell extract. The association constants per binding site for intact, monomeric, and half-monomeric COU-1 were by competitive inhibition assays estimated at 1.5 × 108 M−1 , 3.1 × 108 M−l and 4.0 × 106 M−1 , respectively. The reduction of human IgM MAbs to IgMm and IgMI½m fragments may facilitate the tumor localization when these are used in the diagnosis and therapy of cancer patients. Show more

Keywords: human monoclonal antibody, fragment, monomeric, half-monomeric, antigen-binding, colorectal cancer

DOI: 10.3233/HAB-1993-4207

Citation: Human Antibodies, vol. 4, no. 2, pp. 86-93, 1993