Christopher L. Reading

Recombinant monoclonal antibodies

Recombinant antibody technology is a rapidly evolving field that enables the study and improvement of antibody properties by means of genetic engineering. Moreover, the functional expression of antibody fragments in Escherichia coli has formed the basis for antibody library generation and selection, a powerful method to produce human antibodies for therapy. Because in vitro-generated antibodies offer various advantages over traditionally produced monoclonal antibodies, such molecules are now increasingly used for standard immunological assays. This chapter will give a short review on how recombinant antibodies are generally be produced and engineered, and how typical immunoassays are performed.

In vivo and in vitro properties of malignant variants of RAW117 metastatic murine lymphoma/lymphosarcoma.

Using the RAW117 lymphoma/lymphosarcoma system syngeneic to Balb/c strain mice, variant sublines have been selected for enhanced blood-borne liver colonization in vivo or for lack of binding to immobilized lectins in vitro. The kinetic organ distributions of intravenously injected, 3H-thymidine-labelled RAW117 parental cells and a subline sequentially selected ten times for enhanced liver colonization were similar, suggesting that the differences in malignancy between these two cell lines were not due to dramatic differences in organ localization properties. Examination of the malignant properties of the selected sublines and cell clones derived from these in immune-impaired animals indicated that host immune status was important in determining the quantity of experimental metastases in this system. Although impairment of T-cell or NK-mediated anti-tumor responses by using 400R 60Co-irradiated or Balb/c nude (nu/nu) mice suggested that certain immunologic responses were not effective in preventing experimental metastasis, impairment of macrophage function with chlorine, silica, trypan blue, carrageenan, cyclosphophamide or pristane were effective and resulted in enhanced malignancy of the parental RAW117 line. In contrast, impairment of macrophage function had little or no effect on the experimental metastatic properties of highly malignant RAW117 sublines or clones. In vitro humoral responses or cell-mediated immunologic assays using lymphoid cells from normal or tumor-bearing hosts failed to demonstrate antibody-mediated or antibody-dependent cell-mediated cytotoxicity (ADCC), T-cell or NK-cell responses against RAW 117 cells. However, poly I : C activated macrophages were more effective against parental RAW117 cells than against a highly metastatic subline in cytolysis and cytostasis assays suggesting that the highly metastatic RAW117 cells can more readily escape macrophage-mediated host defenses.

Procedures for in Vitro Immunization and Monoclonal Antibody Production

In this paper, procedures are described for immunization of mouse spleen cells in culture and their subsequent fusion with myeloma cells to produce antibody-secreting hybridomas, and for screening antibody reactivity with cellular and soluble antigens using a modified enzyme-linked immunosorbent assay (ELISA)1. Using these procedures, we have produced monoclonal antibodies reactive with with small haptens, with highly conserved proteins such as actin and calmodulin (Pardue, et al., 1981)., carbohydrates (Reading, 1981), metastatic murine tumor cells (Miner, et al. 1981), and human leukemic cells (Dicke, et al, 1981). In addition, methods for characterizing monoclonal antibody reactivity with cells and with tissue sections and cytocentrifuge preparations using an immunogold technique, for cell separation based on monoclonal antibody binding using immunogold reagents, and for identification of cell surface antigens using a high-pressure liquid chromatography ELISA assay are described. In addition, a new process for biologically producing bi-functional antibody molecules is discussed. The new process involves production of two separate hybridomas secreting monoclonal antibodies to distinct antigens and their hybridization to form a “quadroma” which secretes monoclonal antibodies which are formed by intercellular recombination of the parental heavy and light chains. The construction of “recombinant monoclonal antibodies” may allow new immunological reagents for diagnosis and therapy.

Detection of human hematopoietic differentiationrelated glycoproteins: The Western enzyme-linked lectin analysis

A technique is introduced (Western enzyme-linked lectin analysis, WELLA) for detecting lectin-reactive cellular glycoproteins after separation on the basis of molecular weight in sodium dodecyl sulfate (SDS) polyacrylamide gels. Lectin-reactive glycoproteins are detected on Western transfers by reaction with lectin-peroxidase conjugates followed by development with hydrogen, peroxide and 4-chloro-1-naphthol which forms a purple-gray precipitate. WELLA is more rapid, more sensitive, and the bands are highly reproducible and better resolved than those obtained, by autoradiography of fluorography.Using this technique, we have detected human differentiation-related glycoproteins on cells of different hematological lineages. Both wheat germ agglutinin-peroxidase (WGA-P) and concanavalin A-peroxidase (ConA-P) detected distinct glycoprotein patterns on isolated peripheral blood platelets, lymphocytes, monocytes, erythrocytes and granulocytes. WGA-P detected numerous similarities between immature myeloid cells isolated from bone marrow and acute myelogenous leukemia cells, including major glycoproteins at 20 and 25 kDa. ConA-P detected a similar pattern of glycoproteins between isolated peripheral blood lymphocytes and T-cell acute lymphoblastic leukemia (T-ALL) cells. The T-ALL cells, however, had a major 200 kDa glycoprotein not present on lymphocytes. WGA-P also showed nearly identical patterns between the lymphocytes and the T-ALL cells, but detected prominent 200 and 250 kDa glycoproteins on the T-ALL cells which were absent from the lymphocytes. We have also detected polymorphic differences in the glycoproteins on lymphocytes from normal donors in the range of 95–100 kDa using ConA-P.