Human Antibodies - Volume 7, issue 1

Authors: Xiang, Jim | Qi, Yumin | Cook, Dan | Moyana, Terence

Article Type: Research Article

Abstract: The construction, synthesis and expression of a genetically engineered bifunctional antibody/cytokine fusion protein is described. To target IFN-τ to tumor cells, recombinant antibody techniques were used to construct a RM4/IFN-τ fusion protein containing the chimeric anti-tumor F(ab′)2 (RM4) and the IFN-τ moiety. The recombinant cDNA of IFN-τ was linked to 3 prime end of the chimeric heavy-chain gene fragment (M4) containing the VH , the CHI and the hinge region to form the fused heavy-chain gene fragment M4-IFN-τ. Transfection of the M4-IFN-τ gene fragment into a myeloma derived cell line VK CK which produced the chimeric light-chain …of the same antibody, allowed the transfectant secreting the bifunctional fusion protein RM4/IFN-τ. The RM4 /IFN-τ was purified by the affinity chromatography. Our data showed that the RM4/IFN-τ retained the TAG72 antigen-binding reactivity as well as the IFN-τ activity as measured in ELISA, FACS analysis of cell-surface TAG72 expression, immunohistochemical study, and up-regulation of cell-surface expression of CEA, HLA class I and class II antigens. Therefore, the bifunctional fusion protein RM4/IFN-τ may prove to be useful in targeting biological effects of the IFN-τ to tumor cells and in this way to stimulate the immune destruction of tumor cells. Show more

Keywords: Fusion protein, anti-TAG72 antibody, IFN-τ

DOI: 10.3233/HAB-1996-7101

Citation: Human Antibodies, vol. 7, no. 1, pp. 2-10, 1996

Authors: Vogel, Monique | Lai, Lilin | Rudolf, Michael P. | Curcio-Vonlanthen, Véronique | Miescher, Sylvia | Stadler, Beda M.

Article Type: Research Article

Abstract: Amino acid sequence homology between tetanus toxoid, a common vaccine and melittin, a bee venom allergen provided us with two antigen models for studying the production of cross reactive antibodies after immunisation. Analysis of the serum of an atopic patient recently boosted with tetanus toxoid revealed the presence of anti-melittin and anti-tetanus antibodies. From this donor a phage display Fab library was constructed five days after vaccination. Screening of this library either with melittin alone or with tetanus toxoid followed by melittin allowed the isolation of Fab fragments specific to melittin but also Fab fragments which cross react with melittin …and tetanus toxoid. Amino acid analysis revealed diversity in the heavy and the light Ig chains of the melittin specific and of the cross reactive clones. Interestingly we found that the light chain recognised melittin whereas the heavy chain preferentially bound to tetanus toxoid suggesting that cross reactivity may be due to the different binding specificities of the individualIg chains. Show more

Keywords: In vivo immunisation, TTd, melittin, phage display, cross reactive antibody

DOI: 10.3233/HAB-1996-7102

Citation: Human Antibodies, vol. 7, no. 1, pp. 11-20, 1996

Authors: Qi, Yumin | Moyana, Terence | Chent, Vue | Xiang, Jim

Article Type: Research Article

Abstract: Our previous study showed that the injection of mouse myeloma VK CK /RM4-IFN-τ cells secreting the fusion protein RM4/IFN-τ to syngeneic BALB/c mice resulted in tumor regression in 70% of mice after tumor inoculation. In this study, the VK CK /RM4-IFN-τ cell line was used to characterize the protective immunity subsequent to tumor inoculation. Our histologic findings demonstrated that, in the primary response to VK CK RM4-IFN-τ inoculation, tumor regression is associated with macrophage infiltration. This macrophage-dominated regression further leads to a protective immunity against the 2nd challenge of parental VK CK tumor cells. FACS analysis and chromium release …assays showed that the majority of T lymphocytes that mediated this anti-tumor immunity were CD8+ cytotoxic T lymphocytes (CTLs). Our animal studies further showed that the VK CK /RM4-IFN-τ cells were able to grow as aggressively as the parental VK CK cells in T lymphocyte deficient nude mice. The protective immunity started 7 days, became complete 10 days following and lasted up to at least 12 months subsequent to the tumor inoculation. The adoptive transfer of T lymphocyte-enriched spleen cells or CTLs also conferred significant protection against tumor growth of parental VK CK cells (p < 0.01). These data thus support the notion that genetically engineered tumor cells secreting IFN-τ may have potential use as tumor vaccines in preventing the development of tumor recurrence and/or metastases following the surgical removal of the primary tumors. Show more

Keywords: Fusion protein, macrophage, CD8+ T lymphocyte, anti-tumor immunity

DOI: 10.3233/HAB-1996-7103

Citation: Human Antibodies, vol. 7, no. 1, pp. 21-26, 1996

Authors: Shoji, Masahiro | Kawamoto, Seiji | Seki, Kiyohiko | Teruya, Kiichiro | Setoguchi, Yuko | Mochizuki, Katsumi | Kato, Masatoshi | Hashizume, Shuichi | Hanagiri, Takeshi | Yoshimatsu, Takashi | Nakanishi, Kozo | Yasumoto, Kosei | Nagashima, Akira | Nakahashi, Hisashi | Suzuki, Tatsuo | Imai, Tadaaki | Shirahata, Sanetaka | Nomoto, Kikuo | Murakami, Hiroki

Article Type: Research Article

Abstract: Human monoclonal antibody (hMAb) AE6F4 has been shown to be potentially useful for immunocytological detection of lung cancer cells in sputum. By recombinant DNA technology, IgM type hMAb AE6F4 was switched to IgG. The IgG mimic recombinant AE6F4 antibody expression plasmid was assembled using the antibody heavy chain gene, which ligated the gene encoding VH and CH 1 (μ) domains of hMAb AE6F4 heavy chain to the gene encoding CH 2(γ 1) and CH 3(γ1) domains of human IgG heavy chain, and the antibody light chain gene of hMAb AE6F4. The recombinant antibody expressed by baby hamster kidney (BHK) …– 21 cells showed molecular size equivalence to IgG, and consisted of human μ–γ hybrid heavy and κ light chains. The immunological specificity of the recombinant antibody was the same as that of hMAb AE6F4 by immunoblotting analysis to the 14–3–3 protein, the putative antigen of hMAb AE6F4, and by immunohistochemical and immunocytological analyses using tissue sections and sputa of lung cancer patients. The transfected BHK-21 cells produced the recombinant antibody persistently and the productivity was greater than 20 times that by human-human hybridoma producing hMAb AE6F4. Show more

Keywords: Human monoclonal antibody, recombinant antibody, lung cancer, baby hamster kidney cells, diagnostics

DOI: 10.3233/HAB-1996-7104

Citation: Human Antibodies, vol. 7, no. 1, pp. 27-36, 1996

Authors: Vollmers, H. Peter | Wozniak, Ewa | Stepien-Bötsch, Ewa | Zimmermann, Ulrich | Müller-Hermelink, Hans-Konrad

Article Type: Research Article

Abstract: We describe in this paper the rapid and cheap purification of human immunoglobulin M from hybridoma supernatant of mass culture. The method consists of two steps: 1. concentration of supernatant by ultrafiltration and 2. dialysis against distilled water, pH 6.4. To produce the supernatant, the hybridomas are grown in RPM1 media with fetal calf serum (10% FCS) in 250 ml flasks under normal tissue culture conditions. More than 14 mg IgM can be nearly selectively purified within 6 h from 5 l of antibody containing hybridoma supernatant. Most of the IgM molecules stay in a penta- and monomeric form and …are positive in physiological and immunohistochemical studies. Show more

Keywords: Human immunoglobulin M, immunoglobulin mass culture, water dialysis, pure human IgM

DOI: 10.3233/HAB-1996-7105

Citation: Human Antibodies, vol. 7, no. 1, pp. 37-41, 1996

Authors: Kamiya, Toshio

Article Type: Research Article

Abstract: Human immunoglobulin E (IgE) contains a potential recognition sequence for thrombin protease cleavage at N-terminal end of Cε3 domain responsible for binding to α chain of high affinity Fc receptor for IgE (FcεRICα, but it remains unknown for the enzyme susceptibility. The human Fc fragments of IgE (IgE-Fc), consisting of Cε2'(Ala329)-Cε3-Cε4 chains (Fc') and ofCε2'(Thr315-Ala329)-Cε3-Cε4 chains (F(c')2 ), were expressed in the mammalian COS and Chinese hamster ovary (CHO) cells by placing the /gE-Fc cDNA under the control of the cytomegalovirus (CMV) promoter. Under nonreducing condition F(c')2 was not cleaved by thrombin protease as well as native IgE. Neither …treatment of F(c')2 with final 0.1% 2-mercaptoethanol at boiling point for 5 min, with a sulfhydryl-reactive biotin derivative, by which monomers in COS-derived F(c'h)2 preparations were biotinylated at Cys328, nor with neuraminidase, affected the accessibility to the enzyme. These results suggested that F(c'h)2 , either dimeric or monomeric, had compact conformations. Show more

Keywords: Biotinylation, desialylation, IgE-Fc, thrombin susceptibility

DOI: 10.3233/HAB-1996-7106

Citation: Human Antibodies, vol. 7, no. 1, pp. 42-47, 1996