Human Antibodies - Volume 2, issue 3

Authors: Mochizuki, Katsumi | Kato, Masatoshi | Sato, Susumu | Hashizume, Shuichi | Murakami, Hiroki | Nomoto, Kikuo

Article Type: Research Article

Abstract: A human monoclonal antibody HB4C5, which is known to be associated with lung cancers, was characterized. Two kinds of light chains with 30-kD and 32-kD molecular mass were found to comprise the molecule of this IgM antibody. Various combinations of the two light chains could be contained in this IgM antibody molecule, which might have resulted in the heterogeneity of this antibody with different reactivities. The presence of the 30-kD light chain was indispensable for the antibody activity of cancer recognition. Monomeric 180-kD units of the antibody, having 32-kD light chains exclusively, which showed marginal activity of cancer recognition, could …be removed by immunosorbent column chromatography with histone H2B–Sepharose 4B. F(ab')2 of the HB4C5 antibody containing the active 30-kD component as the dominant light-chain constituent could be purified by hydroxyapatite chromatography by HPLC, facilitating clinical applications of this lung cancer-associated monoclonal antibody of human origin. Show more

Keywords: human monoclonal antibody, IgM antibody, lung cancer, heterogeneity, 180-kD, monomeric unit, F(ab')2

DOI: 10.3233/HAB-1991-2301

Citation: Human Antibodies, vol. 2, no. 3, pp. 116-123, 1991

Authors: Maeda, Hiroaki | Matsushita, Shuzo | Eda, Yasuyuki | Kimachi, Kazuhiko | Tokiyoshi, Sachio | Bendig, Mary M.

Article Type: Research Article

Abstract: Mouse monoclonal antibody (mAb) 0.5β binds to the envelope protein gp120 of human immunodeficiency virus (HIV) and neutralizes infection by HIV in vitro. Mouse mAb 0.5β, therefore, has potential as a therapeutic agent for the prevention and treatment of acquired immunodeficiency syndrome (AIDS). Since mouse mAbs are highly immunogenic in humans, efforts are being made to humanize mouse mAbs that are being considered for use in humans. This report describes the design, construction, and expression of reshaped human 0.5β antibodies. In these antibodies, the entire constant (C) regions were derived from human sequences. The variable (V) regions were derived from …human framework regions (FRs) and mouse 0.5β complementarity determining regions (CDRs). One version of reshaped human 0.5β light (L) chain and six versions of reshaped human 0.5β heavy (H) chain were made and tested. Following transient expression in cos cells, all of the constructions were capable of producing humanlike antibody. Three of the H chain constructions (RHc, RHe, and RHf), when co-expressed with the L chain construction (RL), produced reshaped human antibody capable of binding to the epitope on gp120 recognized by mouse 0.5β mAb. The best version (RL + RHe) of reshaped human 0.5β antibody had both binding affinity and neutralizing activity that were within twofold that of the mouse or chimeric 0.5β antibody. Show more

Keywords: antibody engineering, human immunodeficiency virus, chimeric antibody

DOI: 10.3233/HAB-1991-2302

Citation: Human Antibodies, vol. 2, no. 3, pp. 124-134, 1991

Authors: Ditzel, Henrik | Erb, Karin | Borup-Christensen, Per | Nielsen, Bjarne | Jensenius, Jens Chr.

Article Type: Research Article

Abstract: Preliminary investigations suggested the importance of an evaluation of different tissue preparation methods frequently used for immunohistochemical analysis of human or murine monoclonal antibodies on human tissue. Colon adenocarcinomas and adjacent morphologically normal colon epithelia were analyzed with an indirect immunoperoxidase technique. Duplicate tissue specimens were (1) snap frozen and fixed in acetone, (2) formalin fixed and paraffin embedded, with or (3) without ensuing treatment with pronase, or (4) alcohol fixed and paraffin embedded. Three different human monoclonal anti-colon cancer IgM antibodies, COU-1, D4213, and F10279, were used in the present study. Endogenous immunoglobulin and the secretory-component-mediated IgM binding were …blocked on frozen sections with Fab' anti-IgM and anti-SC antibody. Bound monoclonal antibody was detected with horseradish peroxidase-anti-IgM. COU-1 was found to stain frozen sections of all 25 cancer and adjacent normal colon epithelia. In contrast, on formalin-fixed, paraffin-embedded tissue, only 80% (20/25) of the colon cancer and 44% (11/25) of the adjacent normal colon epithelia were positive. After treatment of the formalin-fixed sections with pronase, all cancers and normal adjacent epithelia were stained, but the cancer cells were more intensely stained than the normal colon epithelial cells. On alcohol-fixed tissues, intense staining was found in all the colon carcinomas analyzed, whereas no staining was found of the adjacent normal colon epithelia, except for a few cells in some of the sections investigated. Antibody F10279 stained none of the frozen, acetone-fixed tissues or the alcohol-fixed and paraffin-embedded tissues analyzed, whereas it stained 71% (5/7) of the formalin-fixed adenocarcinomas and 43% (3/7) of the adjacent normal epithelia. Pronase treatment enhanced the staining intensity. Antibody D4213 showed similar staining of all cancers and normal colon epithelia analyzed with all the four preparation techniques. Procedures for the fixation and processing of human tissue for the immunohistochemical screening of human monoclonal antibodies are critical. Our results demonstrate the necessity of examining several tissue preparation techniques for the analysis of the tissue reactivity of monoclonal antibodies, and indicate that alcohol fixation may advantageously be included. Destruction or masking of antigens, unspecific binding of antibodies, or other technical problems may lead to erroneous conclusions concerning the antigen distribution or the specificity or selectivity of the antibody. Show more

Keywords: human monoclonal antibodies, immunohistochemistry, tissue fixation, COU-1, colon cancer

DOI: 10.3233/HAB-1991-2303

Citation: Human Antibodies, vol. 2, no. 3, pp. 135-141, 1991

Authors: Hashizume, S. | Kamei, M. | Mochizuki, K. | Sato, S. | Kuroda, K. | Kato, M. | Yasumoto, K. | Nakahashi, H. | Hirose, H. | Tai, H. | Okano, H. | Nomoto, K. | Murakami, H.

Article Type: Research Article

Abstract: Cytochrome c from various sources, such as Candida krusei , yeast, horse, and cattle, was found to be recognized by human monoclonal antibody HB4C5 specific to lung cancer. Therefore, the cytochrome c was applied to the measurement of antibody amount in patient sera with a similar reactivity to the antibody HB4C5 for serodiagnosis of cancer. The cytochrome c from Candida krusei was most valuable for the serodiagnosis of various cancers, and the yeast cytochrome c was also useful. However, horse and bovine cytochrome c did not react with antibody of the cancer patients. By …using Candida cytochrome c , lung, bile duct, esophagus, and liver cancers were detected at high rates of more than 50%. In the case of lung cancer, the detection rates of small-cell, squamous, large-cell and adenocarcinoma were 78%, 63%, 100%, and 34%, respectively. The rate for small-cell carcinoma was higher than that with the currently used NSE assay system, and the rate for squamous carcinoma was comparable to that with the SCC assay system, although the system using cytochrome c did not show similar reactivity to that with the SCC system. Furthermore, lung cancer was detected at early stages by using cytochrome c , and even in the case of adenocarcinoma, the rate at early stages with the cytochrome c system was higher than that with the CEA assay system. On the other hand, false positive rates of benign diseases and normal were low–8% and 2%, respectively. Show more

Keywords: serodiagnosis, cytochrome c, Candida krusei, lung cancer, human monoclonal antibody

DOI: 10.3233/HAB-1991-2304

Citation: Human Antibodies, vol. 2, no. 3, pp. 142-147, 1991

Authors: Simonsson, Ann Catrin | Larrick, James W. | Borrebaeck, Carl A.K.

Article Type: Research Article

Abstract: The kinetics of lymphokine-specific DNA transcription during in vitro immunization of human peripheral blood lymphocytes and splenocytes were studied using the polymerase chain reaction. The levels of specific mRNA were shown to be downregulated by cytolytic L-leucyl-leucine methyl ester-sensitive lymphocytes. In in vitro immunizations using L-leucyl-leucine methyl ester-treated human PBL or splenocytes, the lymphokine mRNA expression pattern indicated an active gene transcription during the entire stimulation period, especially for the IL-2 and IL-5 genes. Transcription of IL-6 and TNFβ started on day 4, whereas IFNγ mRNA reached its maximum level on day 4. In vitro immunizations of cells not treated …with L-leucyl-leucine methyl ester revealed a transient transcription of lymphokine DNA that was declining already after day 2. Exogenously added recombinant IL-2, IL-4, and IL-6 all exhibited a positive immunoregulatory effect on Ig secretion, whereas IL-5 was not found to have any effect on immunoglobulin secretion during the in vitro culture. These results present the first information useful for designing in vitro immunization systems based on recombinant lymphokines and antisense DNA for gene regulation. Show more

Keywords: in vitro immunization, gene mapping, lymphokine mRNA

DOI: 10.3233/HAB-1991-2305

Citation: Human Antibodies, vol. 2, no. 3, pp. 148-154, 1991

Authors: Perkins, Susan | Zimmermann, Ulrich | Foung, Steven K.H.

Article Type: Research Article

Abstract: Hypoosmolar conditions have permitted the development of electrofusion techniques capable of producing human hybridomas from as few as 105 B cells. A hybridoma formation efficiency of one hybrid for each 125 input B cells has been achieved with Epstein-Barr virus-activated B cells and mouse–human heteromyelomas. This is at least 100-fold higher in efficiency than with polyethylene glycol-induced cell fusion, as well as a 50- to 100-fold decrease in the required number of human B cells. The ability to fuse a small number of input B cells should lead to a greater success rate in immortalizing the rare antigen-specific B …cells. The critical parameters include fusion voltages, the composition and number of wash steps used in cell preparation, the composition and duration of exposure to hypoosmolar fusion medium, fusion ratio, plating density, the use of growth medium without pH indicator, and the use of an irradiated human fibroblast feeder layer. By manipulating these parameters, a high hybrid yield can be achieved with different mouse–human heteromyelomas and Epstein-Barr virus–activated B cells. Show more

Keywords: human hybridoma, human monoclonal antibody, electrofusion, hypoosmolar fusion medium

DOI: 10.3233/HAB-1991-2306

Citation: Human Antibodies, vol. 2, no. 3, pp. 155-159, 1991

Authors: Kunicka, Jolanta E. | Fox, Floyd E. | Seki, Hidetoshi | Oleszak, Emilia L. | Platsoucast, Chris D.

Article Type: Research Article

Abstract: With the objective of developing human T-T cell hybrids producing B-cell growth factor, we fused concavalin A–activated T lymphocytes with cells of the lurkat T cell line. The hybrids were selected on the basis of their ability to form colonies in soft agar, whereas the parent Jurkat T cell line did not. T-T cell hybrids were HLA-typed, screened by functional tests, and recloned by limiting dilution. In addition to obtaining B-cell growth factor-producing hybrids, we also obtained certain other T-T cell hybrids (as determined by HLA-typing) producing suppressor factors inhibiting proliferative responses and antibody production by human lymphocytes. Subsequently, a …suppressor factor with similar inhibitory properties was identified in supernatants of the Jurkat T cell line. However, the Jurkat factor exhibited different biochemical and functional properties than the hybridoma-derived suppressor factors. Using two-parameter cell cycle analysis and the metachromatic fluorochrome acridine orange, we found that the hybridoma-derived 160 and 169 suppressor factors arrested phytohemagglutinin-induced proliferation of peripheral blood mononuclear cells in the G0 /G1 phase of the cell cycle, whereas the Jurkat suppressor factor arrested proliferation in the S phase. Incubation of peripheral blood mononuclear cells with the 160, 169, or Jurkat suppressor factors for 24 hr at 37° C, followed by washing, did not alter their cell cycle progression (or RNA content) in response to stimulation with phytohemagglutinin. The hybridoma-derived 160 and 169 suppressor factors and the Jurkat factor inhibited the growth but not the viability of cells from the following human tumor cell lines: A673 sarcoma cell line, SK-LC-6 and SK-LC-14 lung cell lines, SB, Raji, and Daudi lymphoblastoid cell lines, and FARR malignant melanoma cell line. In contrast, it did not affect the growth of murine L1210 cells and FS-4 normal human diploid fibroblasts. The hybridoma-derived 160 suppressor factor was selected to investigate its effect on cell-mediated cytotoxicity: The 160 suppressor factor did not inhibit natural killer cytotoxicity or its augmentation by interferon alpha or interleukin 2 or the generation of lymphokine-activated killer cells. However, this factor partially inhibited the generation of specific T cell-mediated cytotoxicity. Show more

Keywords: human T-T cell hybrids, suppressor factors, cell cycle, inhibition of tumor cell growth

DOI: 10.3233/HAB-1991-2307

Citation: Human Antibodies, vol. 2, no. 3, pp. 160-169, 1991