Human Antibodies - Volume 8, issue 1

Article Type: Editorial

DOI: 10.3233/HAB-1997-8101

Citation: Human Antibodies, vol. 8, no. 1, pp. 2-2, 1997

Authors: Léger, Olivier J.P. | Yednock, Ted A. | Tanner, Laura | Horner, Heidi C. | Hines, Dawn K. | Keen, Simon | Saldanha, José | Jones, S. Tarran | Fritz, Lawrence C. | Bendig, Mary M.

Article Type: Research Article

Abstract: α4β1 integrin (VLA-4) is crucialfor the adhesion of leukocytes to human vascular cell adhesion molecule-1 (VCAM-1) on iriflamed endothelium. This cell adhesion event is the first step in leukocyte extravasation across the blood-brain barrier in iriflammatory diseases of the central nervous system (CNS) such as experimental autoimmune encephalomyelitis (EAE). Prevention of leukocyte infiltration by antibodies against the α4 integrin, which block the α4β1 integrin/VCAM-1 interaction, have been shown to suppress clinical and pathological features of EAE. In this study, two mouse monoclonal antibodies (MAb) directed against human α4 integrin were analyzed in vitro for their ability to block the interaction …of leukocytes with VCAM-1 under different assay conditions. The best blocking MAb, AN100226m, was humanized by complementarily-determining region grafting, associated with human C regions and expressed. We found that modification of two structural determinants (H27 and H29) for the heavy chain CDRI loop in one hand, and modification of framework amino acid H38, H40 and H44 in the other hand, had no effect on antigen binding. In contrast, modification of a structural determinant (H71) for the heavy chain CDR2 loop resulted in loss of binding. The humanized antibody, AN100226, was equivalent to the murine antibody, AN100226m, in binding to α4β1 integrin and in blocking cell adhesion. More importantly, AN100226 was as effective as AN100226m in the reversal of active EAE in guinea pigs and thus may be useful in the treatment of autoimmune diseases such as multiple sclerosis. AN100226 is currently in phase II clinical trials in the UK for the treatment of multiple sclerosis exacerbations. Show more

Keywords: Adhesion molecules, humanization, CDR-grafting, antibodies, α4β1 integrin, experimental allergic encephalomyelitis

DOI: 10.3233/HAB-1997-8102

Citation: Human Antibodies, vol. 8, no. 1, pp. 3-16, 1997

Authors: Klein, Philippe | Béliard, Roland | Glacet, Arnaud | Moitel, Sébastien | Ducrot, Thierry | Bourel, Dominique

Article Type: Research Article

Abstract: Therapeutic use of human monoclonal antibodies has so far been hampered by their poor availability. Recent developments in recombinant DNA technologies are expected to fill this gap: the variable and constant sequences of antibodies can be selected independently and then subsequently joined to express whole antibodies. We assessed here the potential of this methodology to obtain novel anti-D antibodies with improved biological characteristics. The sequences coding for heavy and light chains of two anti-Rh(D) monoclonal antibodies (one IgGI and one IgG3) were isolated and co-expressed into murine myeloma cell P3X63.Ag8.653, either directly. (parental antibodies) or after exchange of constant heavy …chain sequences (class-switched antibodies). Parental antibodies produced either by transfectomas or by hybridomas behaved similarly in analysis of biochemical, binding and effector properties. Class-switched antibodies displayed altered functional properties over parental antibodies. Of particular interest, one of them showed improved phagocytosis potencies over both parental antibodies. Additionally, these results indicate that functional properties do not always simply reflect the addition of properties of constant and variable parts but that interactions between constant and variable regions may interfere. Show more

Keywords: Human monoclonal antibodies, recombinant antibodies, anti-D, transfectomas, mammalian expression, effector functions

DOI: 10.3233/HAB-1997-8103

Citation: Human Antibodies, vol. 8, no. 1, pp. 17-25, 1997

Authors: Gore, Milind M. | Singhania, Smita S. | Basu, Atanu | Banerjee, Kalyan

Article Type: Research Article

Abstract: B cell growth and differentiation into immunoglobulin secreting cells is controlled by various cytokines and cell to cell contact with T cells. Fusion partner for human hybridoma therefore should accommodate all or some of these signaling systems to overcome the unique situation of MHC incompatibility, need for specific growth factors simultaneously taking into consideration the downstream processing of the product for the clinical use. We have thus directed our efforts towards the development of a fusion partner which would not need Epstein-Barr virus transformation of B cells prior to fusion. A nontransforming mitogen, formalinized Staphylococcus aureus (FSTA) was used for …stimulating human B cells. Successful production of human JgM monoclonal antibody was achieved by incorporating Jurkat-4 cells in existing mouse human heterohybrid through fusion of these cells followed by fusion with human B cells. To accommodate chromosomes of both T and B cells after fusion, human myeloid precursor cells KG1a, and to incorporate T cell, HuT78 cells were fused. CD34+ and CD4+ hybrid of KG1a and HuT 78 cells – 434 AM – when used as fusion partner could allow secretion of MAbs, however growth potential was low. SP2/0 cells were then incorporated in 434 AM cells to give myeloma environment to fused human B cells. Rabies virus neutralizing human IgG MAb secreting clone was generated by fusing FSTA stimulated human B cells with this fusion partner. Show more

Keywords: Human MAb, fusion partner development, T cell fusion, myeloid precursor, rabies virus

DOI: 10.3233/HAB-1997-8104

Citation: Human Antibodies, vol. 8, no. 1, pp. 26-32, 1997

Authors: Haedicke, Wolfgang | Knörr, Constanze | Müller-Hermelink, Hans Konrad | Greiner, Axel

Article Type: Research Article

Abstract: The generation of human antibodies derived from extranodal MALT type B-cell lymphomas allows to evaluate steps in their pathogenesis as well as to establish potential immunological therapies. Intraclonal diversity and the existence of bystander nonmalignant B-cells outline the need for reliable identification of the tumor immunoglobulin representing hybridomas. Human heterohybridomas were generated from five cases of MALT type B-cell lymphomas (4 low grade, one high grade) by the fusion of lymphoma B-cells with the murine myeloma cell line NSO and tested for isotype identity with the tumor. RT-PCR using VH Frl/JH primers was performed with RNA of tumors and hybridomas …that share the same isotype with the tumor. PCR-products were sequenced directly. In each case lymphomas were hybridized with a comparable fusion efficiency. DNA sequence analysis of the immunoglobulin heavy chain identified one or more hybridomas derived from the tumor. However some other hybridomas which share the same isotype with the tumor, maybe different in their VH family or their sequence. Hybridomas can be used as a tool for the research on the MALT lymphoma immunoglobulin receptor. For the identification of tumor immunoglobulin, secreting hybridomas sequencing and the check of molecular identity is indispensable after isotype determination. Show more

Keywords: Human antibody, MALT lymphoma, hybridoma, immunoglobulin heavy chain, isotype

DOI: 10.3233/HAB-1997-8105

Citation: Human Antibodies, vol. 8, no. 1, pp. 33-36, 1997

Authors: Sodoyer, Régis | Peubez, Isabelle | Pion, Corinne | Dubayle, Joseline | Jacquemot, Paul | Geoffroy, Frederique | Even, Jos | Aujame, Luc

Article Type: Research Article

Abstract: Very large ‘naïve’ human antibody repertoires have been obtained from RT-PCR cloned VH and VL variable regions. They are used as starting material for the assembly of medium sized combinatorial libraries or so called multicombinatorial libraries. In nonimmunized individuals immunoglobulin messenger RNAs are poorly expressed, which can be a serious limitation for cloning efficiency. To overcome this problem two complementary strategies have been used: a nonspecific polyclonal activation of B cells, and a secondary PCR amplification technique to ensure recovery of Ig messengers in large amount and without introducing any bias.

Keywords: Phage display, combinatorial library, F(ab) fragment, human naïve repertoire

DOI: 10.3233/HAB-1997-8106

Citation: Human Antibodies, vol. 8, no. 1, pp. 37-42, 1997

Authors: Tsuchiyama, Linda | Kieran, Jane | Boyle, Petra | Wetzel, Gayle Delmonte

Article Type: Research Article

Abstract: For human B lymphocytes, Epstein-Barr virus (EBV) is a polyclonal activator, inducing both proliferation and Ig secretion. It is also a transforming virus capable of generating immortalized B cell lines. These early and late functions of EBV are not apparently connected. The receptor for EBV CD21, also serves as a receptor for some complement components and is called CR2. This molecule associates with CD19 and TAPA-1 on the surface of B cells. This complex is involved in signaling B cells and participates in many responses. We have observed that simultaneous ligation of CD40 and the CD21 complex, by exposure to …anti-CD40 MAbs and EBV enhances both the short-term proliferation as well as the long-term transformation rate of human B lymphocytes. B cell proliferation shows synergy between anti-CD40 MAb and EBV CD19 also appears to be involved in the synergistic activation of B cells through CD40 and CD21, since ligation of CD19 with anti-CD19 MAbs, either prior to or concomitant with exposure to anti-CD40 and EBV markedly inhibits both proliferation and subsequent B cell transformation. These observations do not elucidate the mechanisms of B cell transformation employed by EBV but they do suggest a relationship between early proliferation and later transformation induced by the virus. Anti-CD40 enhances both these effects and anti-CD19 is capable of inhibiting both. Show more

Keywords: EBY, CD40, transformation, human B-lymphocytes

DOI: 10.3233/HAB-1997-8107

Citation: Human Antibodies, vol. 8, no. 1, pp. 43-47, 1997