PCR cloning and sequencing of a CD3-specific monoclonal antibody.
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Amino Acid Sequence↗
Biomedical subjects
H Heyligen
Publications and source records attributed to H Heyligen.
Mono- and bispecific single-chain antibody fragments for cancer therapy.
Especially when dealing with solid cancers, single-chain antibody fragments (scFvs) have a lot of advantages. Due to their small size (27 kDa), these proteins clear more rapidly from the blood, and penetrate faster and deeper into tissues, than whole antibodies. Furthermore, the lack of constant regions ensures that they are not retained in tissues such as the liver and kidney. This reduces possible toxic side-effects. Single-chain construction is normally done by polymerase chain reaction (PCR). To decrease the overall cost of oligonucleotide primer synthesis, time-consuming primer design, multiple PCR reactions and individual PCR optimization, we designed a universal single-step overlap extension PCR protocol using hybridoma cDNA as a template. To overcome the lack of effector function, bispecific scFvs, consisting of an scFv produced against a tumour-associated antigen fused to a T cell marker-specific scFv, are being created, starting from already assembled scFv, by means of two additional PCR reactions. In this paper we describe both PCR methods that were successfully used to create scFvs against the human transferrin receptor, the human interleukin-2 receptor, the human CD3 molecule, a breast tumour-associated antigen and an anti-transferrin-anti-CD3 bispecific scFv.
Antibodies, Bispecific↗
Immunotherapy for cancer: construction, expression and functional characterization of chimeric antibodies.
Monoclonal antibodies (Mabs) are a potential key component for the treatment of cancer, because of their specificity and multiple effector functions. Hybridoma technology and progress in genetic engineering made it possible to customize antibody molecules, rendering them more suitable for selective application. A widely used technique is the construction of mouse-human hybrid molecules by recombinant DNA techniques. These so-called chimeric antibodies contain the murine variable (V) regions fused to the human constant (C) regions. In this report, a general approach is described for the production of chimeric antibodies. The gene segments encoding the murine variable heavy and light chain are isolated by the polymerase chain reaction and cloned into expression vectors containing the human gamma 1 heavy chain gene and the human K light chain gene, respectively. Subsequently, these constructs are transfected into a non-Ig-producing murine hybridoma, eg SP2/0 cells. The in vitro study of the functional characteristics and biological properties of the thus obtained chimeric antibodies are discussed.
Antibodies, Monoclonal↗
Development and functional characterization of a murine/human chimeric antibody with specificity for the human interleukin-2 receptor.
A murine/human chimeric antibody, with specificity for the human interleukin-2 receptor, was developed by genetic engineering. For this purpose, the light and heavy chain variable region exons encoding the murine monoclonal antibody 2C8 were isolated and inserted into expression vectors containing the human kappa and gamma-1 constant regions. After transfection by electroporation of the chimeric genes into murine Sp 2/0 hybridoma cells, transfectomas secreting the complete chimeric antibody were selected. The chimeric antibody has similar binding properties as the original murine antibody. The in vitro cytotoxic effects of the murine and the chimeric antibodies were compared.
Amino Acid Sequence↗
Antibody binding profile of purified and cell-bound CD26. Designation of BT5/9 and TA5.9 to the CD26 cluster.
The CD26 activation antigen (Ag) which is expressed on a subpopulation of human T cells has been characterized as dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5). In this paper, we describe the antibody binding profile of CD26/DPP IV, purified from human peripheral blood lymphocytes. The purified molecule binds to the anti-Ta1, anti-1F7 and anti-134-2C2 monoclonal antibodies (mAb), reported to react with cell-bound CD26 Ag. Among unclustered mAb recognizing T cell antigens, two, anti-BT5/9 and anti-TA5.9 were found to react with purified and cell-bound CD26 Ag. The classification of the BT5/9 Ag, the functional properties of the BT5/9+ T cell subset, as well as the in vivo effect of anti-BT5/9 mAb administration, are re-interpreted in the light of its specificity. Applying the anti-TA5.9 mAb in three color FACS analyses, we demonstrated that CD26+bright cells co-express CD45RO but not HLA-DR and CD38.
Antibodies, Monoclonal↗
Decreased expression of the memory marker CD26 on both CD4+ and CD8+ T lymphocytes of HIV-infected subjects.
Using a novel anti-CD26 (or anti-dipeptidyl peptidase IV) monoclonal antibody, we showed that the absolute numbers and the proportions of T4 and T8 cells expressing CD26 were significantly lower in HIV-infected persons than in controls. The absolute number of CD26+ T4 cells decreased according to disease progression, whereas the number of CD26+ T8 cells was low throughout all clinical stages. These trends were similar in CD26 dim and bright positive T-cell subsets. In both controls and HIV-positive subjects, the CD26 bright positive T cells were restricted to the CD45RO+ subset and preferentially co-expressed CD25 but largely lacked HLA-DR and CD38. Recall antigen-responsive cells from seronegative individuals were shown to co-express CD26 and CD45RO. The deficient CD26 expression on T8 cells from HIV-infected subjects could be normally upregulated after in vitro stimulation. In contrast to decreased T-cell-bound CD26, the enzymatic activity of plasma CD26/dipeptidyl peptidase IV was unchanged in HIV-infected patients compared with controls. We conclude that HIV infection leads to a deficient in vivo co-expression of CD26 bright and CD45RO on T cells. We speculate that this deficiency might play a part in the decrease of immunological memory during HIV infection.
ADP-ribosyl Cyclase↗
Human B cell lines secreting IgM antibody specific for myelin basic protein.
In this study we describe for the first time the production of stable human B cell lines and clones that secrete IgM antibody specific for human myelin basic protein. The technique based on limiting dilutions of Epstein-Barr virus (EBV)-transformed peripheral B cells from patients with multiple sclerosis precluded the need for preselecting or stimulating antigen-specific B cells. Most of the cell lines were stable for at least 6 months in continuous culture and produced 5-12 micrograms/ml antibody after 2 weeks in culture. The myelin basic protein (MBP)-specific B cells were surface IgM positive, and occurred with a frequency of approximately 1/2500 mononuclear cells in peripheral blood. The successful selection and quantitation of specific B cell clones described here suggests that this technique is well suited for evaluating B cell responses to known and suspected antigens and autoantigens.
Antibodies, Anti-Idiotypic↗
Human B cell lines secreting IgM antibody specific for myelin basic protein.
In this study we describe for the first time the production of stable human B cell lines and clones that secrete IgM antibody specific for human myelin basic protein. The technique based on limiting dilutions of Epstein-Barr virus-transformed peripheral B cells from patients with multiple sclerosis, precluded the need for preselecting or stimulating antigen-specific B cells. Most of the cell lines were stable for at least 6 months in continuous culture and produced 5-12 micrograms/ml antibody after 2 weeks in culture. The myelin basic protein-specific B cells were surface IgM positive, and occurred with a frequency of approximately 1/2500 mononuclear cells in peripheral blood. The successful selection and quantitation of specific B cell clones described here suggests that this technique is well suited for evaluating B cell responses to known and suspected antigens and autoantigens.
Antibody Specificity↗
Leu-3+ lymphocytes account for increased CSF cellularity.
Inflammatory conditions of the central nervous system (CNS) are often marked by an increase in lymphocyte number in the cerebrospinal fluid (CSF). In order to determine if changes in CSF cell numbers can alter T-lymphocyte subset composition in CSF or in blood, cell surface markers were evaluated in 25 CSF and paired blood samples from a variety of neurologically affected patients. T-cell subset levels in peripheral blood did not reflect subset levels in paired CSF samples. However, CSF samples with elevated cell numbers (greater than 3 cells/mm3) had significantly increased levels of Leu-3+ T-cells (P less than 0.001), but not Leu-2+ T-cells relative to CSF samples with low cell counts. These data suggest a selective increase in the Leu-3+ T-lymphocyte subset in CSFs with increased cellularity in patients with acute neurologic signs.
Antigens, Differentiation, T-Lymphocyte↗