Human Antibodies - Volume 9, issue 2

Authors: Kawahara, Hiroharu | Maeda-Yamamoto, Mari | Hakamata, Katsuhiro

Article Type: Research Article

Abstract: A new human fusion partner, ICLU-T was established for making human T-T hybridomas. Some 6-thioguanine resistant (6-TGr ) clones were separated from human T cell acute lymphocytic leukemia, PEER. The fastest growing clone of the 6-TGr cells was scaled up and was cultured in serum free medium. The clone that proliferated most quickly in the serum free medium was separated and named ICLU-T. When ICLU-T was fused with human peripheral blood lymphocytes by using only polyethylene glycol, the viability of fused cells was below 20% efficiency was 0.01~0.02 per 105 parent cells. Whereas, the combined use of 1% …and the fusion efficiencies over 0.2 per 105 parent cells on the average. In a total of 34 clones of obtained hybridomas, there were 25 CD2 positive clones. CD4 positive or CD8 positive clones of the CD2 positive clones were 12 and 5 clones, respectively. Further, 4 clones of all hybridomas were CD19 positive, and one clone of these hybridomas was an Ig producer. These obtained hybridomas could proliferate in serum free medium. Show more

Keywords: human T-T hybridomas, lecithin, serum free culture, human fusion partner

DOI: 10.3233/HAB-1999-9201

Citation: Human Antibodies, vol. 9, no. 2, pp. 83-87, 1999

Authors: Kawahara, Hiroharu | Maeda-Yamamoto, Mari | Suzuki, Makiko | Hakamata, Katsuhiro

Article Type: Research Article

Abstract: To investigate human immune responses, it is useful to use cultured human immunological cell lines. Cell fusion is one method to immortalize desired cells. Therefore, we established HAT sensitive human fusion partners, ICLU-B derived from human burkitt lymphoma, ICLU-T from human T cell acute lymphocytic leukemia, ICLU-E from human eosinophilic leukemia, for efficiently making various human immunological hybridomas. When each fusion partner was fused with human peripheral blood lymphocytes or cord blood lymphocytes, their fusion efficiencies were about 1.3, 0.3 and 0.4 clones per 105 fusion partners. Approximately 70% derived from ICLU-B were CD19 positive hybridomas, and approximately 80% …CD4 positive or CD8 positive hybridomas of the CD2 positive were approximately 35% hybridomas derived from ICLU-E were CD2 or CD19 positive hybridomas, phagocytosis was observed in the remaining hybridomas which were both CD2 and CD19 negative. Most obtained hybridomas could proliferate in serum free medium as well as the fusion partners. These results suggested that ICLU-B, -T, -E were useful for making human hybridomas derived from various immunological cells. Show more

Keywords: human immunological hybridomas, serum free medium, human fusion partners, cell fusion

DOI: 10.3233/HAB-1999-9202

Citation: Human Antibodies, vol. 9, no. 2, pp. 89-94, 1999

Authors: Thomas, Myles D. | Clough, Kevin | Melamed, Mark D. | Stevenson, Freda K. | Chapman, Caroline J. | Spellerberg, Myfanwy B. | Potter, Kathleen N | Newton-Bishop, Julia A. | Gregson, Norman | Dawson, Karren

Article Type: Research Article

Abstract: A heterohybridoma cell line producing the human monoclonal antibody (MoAb) MDT.1 has been established. The heavy chain of MoAb MDT.1 is encoded by the VH gene segment V4-34 (previously designated VH 4-21), and the light chain is encoded by the Vκ 1-L12a gene segment, both in germline configuration. MDT.1 has reactivity against lipid A, double- and single-stranded DNA, red blood cell associated i antigen, and ganglioside antigens. In a panel of tumour cell lines, MDT.1 reacted specifically with melanoma cells and other tumour cells of neuroectodermal origin. Cellular recognition appears to be via tumour-associated ganglioside antigens, and may …involve the minimal essential epitope NeuNacα 2→3Galβ 1→-4Glc-. Binding to ganglioside antigen is inhibited by the monoclonal anti-idiotypic antibody 9G4. Since the 9G4 idiotope is located in framework region 1 (FWR1) of V4-34 -encoded antibodies, this region is likely to be involved, either directly or indirectly, in ganglioside binding. The complementarity-determining region 3 (CDR3) of MDT.1 is arginine rich, with five out of 12 residues being arginine and these residues are candidates for interaction with the negatively charged ganglioside. The ability of MoAb MDT.1 to recognise ganglioside antigens is associated with potentially useful anti-tumour activity. Show more

Keywords: human monoclonal antibody, melanoma, ganglioside, autoantibody

DOI: 10.3233/HAB-1999-9203

Citation: Human Antibodies, vol. 9, no. 2, pp. 95-106, 1999

Authors: Sidorova, Ekaterina

Article Type: Research Article

Abstract: Human monoclonal IgM antibodies to synthetic peptide MN-24 corresponding to 303–325 residues of gp 120 HIV were obtained. Antibodies are made synthetic by CD5+B lymphocytes and are polyspecific: they react not only with different short peptides but with some macromolecular antigens as well. Anti-MN-24 antibodies possess antigen-binding activity but do not possess virus neutralizing activity. It is suggested that anti-MN-24 antibodies belong to the natural antibodies.

Keywords: human monoclonal antibody, viral peptides, B cells, natural antibodies

DOI: 10.3233/HAB-1999-9204

Citation: Human Antibodies, vol. 9, no. 2, pp. 107-110, 1999

Authors: Nishimura, Eisaku | Mochizuki, Katsumi | Kato, Masatoshi | Hashizume, Shuichi | Haruta, Hirotaka | Shirahata, Sanetaka | Suzuki, Tatsuo | Nomoto, Kikuo | Kanaya, Kazuko | Kusakabe, Kiyoko

Article Type: Research Article

Abstract: Recombinant λ light chain of lung cancer-reacting human monoclonal antibody HB4C5 was expressed in Escherichia coli . Expression in bacteria ensured the generation of homogeneous light chain species devoid of activity-hampering N-linked glycosylation usually found in the light chain CDR-1 of HB4C5. Molecular engineering was also employed to eliminate the C-terminal two amino acid residues, i.e., Cys and Ser, to prevent the formation of λ light chain dimers which are less reactive than the monomeric form. The λ light chain was overexpressed in E. coli as inclusion bodies, which were solubilized, refolded, and treated with Aeromonas …proteolytica aminopeptidase to remove the N-terminal Met with subsequent natural cyclization of the penultimate Gln residue to pyroglutamate, the same N-terminal end as that of naturally occurring λ light chain in HB4C5. Monomeric recombinant λ light chains, both before and after removal of the N-terminal Met residue, were 40 times more immunoreactive than the parent HB4C5. The immunostaining of lung cancer tissue sections with the recombinant λ light chain indicated cancer-specific reactions to all specimens of adenocarcinoma, squamous cell carcinoma and large cell carcinoma histologies, but did not react with small cell carcinoma. Tumor radioimmunoimaging experiments in LC6 (lung squamous cell carcinoma line) – xenografted nude mice by the i.p. injection of 125 I-labeled recombinant λ light chain and 125 I-labeled human λ light chain control gave tumor-specific and recombinantλ light chain-dependent images on day 5 postinjection, and images were also detectable on day 3. Biodistribution studies with 125 I-labeled recombinant λ light chain demonstrated that the λ light chain could penetrate better into the tumor sites, both at the necrotic and solid parts of the xenograft, as compared to our previous results with 125 I-labeled HB4C5 which could localize to the necrotic part only. These results suggest that the recombinant λ light chain is potentially useful as a lung cancer-targeting vehicle, for such as radioimmunoimaging and radioimmunotherapy, with least possible adverse immunogenic effects. Show more

Keywords: recombinant, light-chain, human monoclonal antibody, lung cancer, radioimmunoimaging, biodistribution

DOI: 10.3233/HAB-1999-9205

Citation: Human Antibodies, vol. 9, no. 2, pp. 111-124, 1999

Authors: George, J. | Aron, A. | Levy, Y. | Gilburd, B. | Ben-David, A. | Renaudineau, Y. | Zonana-Nachach, A. | Youinou, P. | Harats, D. | Shoenfeld, Y.

Article Type: Research Article

Abstract: Patients with chronic renal failure requiring regular hemodialysis are known to be prone to thromboembolic events due to a hypercoagulable state. Vascular access thrombosis (VAT; including thrombosis of the vascular shunt or graft) represents a serious complication and jeopardizes life in these patients. In the current study, conducted on 81 consecutive patients from the Hemodialysis Unit, we have employed ELISA for the estimation of various autoantibody levels (anti-endothelial cell antibodies, anti-cardiolipin, anti-β 2GPI and anti-modified LDL antibodies) and correlated them with the occurrence of thromboembolic events in general, and VAT in particular. We have found that the levels …of antibodies reactive with human umbilical vein endothelial cells (HUVEC) but not with other EC lines (microvascular or EaHy 929) were significantly higher in hemodialysed patients with VAT in comparison with patients with no VAT (p = 0.001 ). A weaker but yet positive correlation was observed between the levels of anti-HUVEC and anti-cardiolipin antibodies and the occurrence of thromboembolic events including deep vein thrombosis, pulmonary infarction, cerbrovascular events and VAT (both p-values equal 0.02). Anticardiolipin antibodies (aCL) were not cross reactive with β 2GPI or with HUVEC. Antibodies to modified LDL, although higher in hemodialyzed patients, did not correlate with thromboembolic events. The results of this study suggest that antibodies to HUVEC may prove as a fairly good marker of VAT in hemodialysis. High levels of aCL are weakly associated with thromboembolic events and antibodies to modified LDL do not correlate with a prothrombotic state. Show more

Keywords: Anti endothelial cell antibodies, hemodialysis, thromboembolic events

DOI: 10.3233/HAB-1999-9206

Citation: Human Antibodies, vol. 9, no. 2, pp. 125-131, 1999

Authors: Smikle, Monica F | Barton, Everard N | St C Morgan, Owen | Luseko, John | Bailey, Valrie E | Williams, Evadne M

Article Type: Research Article

Abstract: The reports of the occurrence of HTLV-1 infection and/or HTLV-1 associated myelopathy (HAM/tropical spastic paraparesis (TSP) in patients with certain organ-specific and nonorgan-specific autoimmune diseases prompted us to assess the relationship between TSP and humoral autoimmunity. Blood samples from 76 TSP patients, 60 asymptomatic HTLV-1 carriers and 100 HTLV-1 seronegative blood donors were examined for the presence of organ-specific and nonorgan-specific autoantibodies, reactive serological tests for syphilis, immunoglobulin and complement concentrations as well as immunecomplexes. High prevalences of autoantibodies (39/76, 51%), reactive serological tests for syphilis (23/76; 30%), hypergammaglobulinaemia (69/76, 90%) and complement fixing immune complexes (44/76, 58%) were found …in the TSP patients. These indicators of immunological disorder were found in statistically significantly lower prevalences in asymptomatic HTLV-1 carriers (12/60, 20%; p < 0.001 ; 6/60, 10%; p < 0.05 ; 32/60, 53%; p < 0.001 and 8/60, 13%; p < 0.001 , respectively) and HTLV-1 seronegative blood donors (8/100, 8%; p < 0.001 ; 3/100, 3%; p < 0.001 ; 15/100, 15%; p < 0.001 and 5/100, 5%; p < 0.001 , respectively). The profiles of autoimmune phenomena observed in the patient and control groups revealed that they were associated with TSP rather than mere HTLV-1 infection and consequently pathogenetic significance. The array of immunological features present in TSP was suggestive of autoimmune disease resulting from immune dysfunction. Studies which explore the possible existence of HTLV-1 induced autoantibodies with specificity for antigens of the spinal cord in TSP might be useful in elucidating its pathogenesis. Show more

DOI: 10.3233/HAB-1999-9207

Citation: Human Antibodies, vol. 9, no. 2, pp. 133-137, 1999