Determinants recognized by murine rheumatoid factors: molecular localization using a panel of mouse myeloma variant immunoglobulins.

The structures recognized by monoclonal anti-IgG1 rheumatoid factors (RF) were localized by testing their reactivity with mutant immunoglobulins carrying gamma 1 chains that lacked either the CH1 or the CH3 domains. While optimal binding was observed in the absence of CH1, deletion of CH3 completely abolished the reactivity of all but one of the 71 monoclonal RF tested. Similar experiments were carried out with IgG2a- and IgG2b-specific RF by using variant immunoglobulins carrying various hybrid gamma 2a-gamma 2b heavy chains. It was found that both the polyclonal RF produced by autoimmune strains, MRL/MpJ-lpr and NZB/BinJ, and most of the monoclonal RF derived from normal strains, BALB/c, C57Bl/6, and 129/Sv, were directed against determinants located in a segment spanning the C-terminal 8 residues of the CH2 domain and the complete CH3 domain.

Pharmacokinetics and immune response of 131I-chimeric mouse/human B72.3 (human gamma 4) monoclonal antibody in humans.

Chimeric B72.3, composed of the V-regions of murine B72.3 and the constant regions of human immunoglobulin G4 heavy and kappa light chain, was administered as a 131I-labeled conjugate to 12 patients with metastatic colon cancer. Seven of these patients had an antibody response after initial infusion, and the immune response was primarily directed to the murine V-region, although a small proportion of the antibody response was directed to topographical epitopes requiring the presence of both murine V-region and human CH-1 and kappa constant regions (neo-epitopes). The pharmacokinetics included a plasma disappearance curve best fit by a two-compartmental model with an alpha t 1/2 of 18 +/- 7 h and a beta t 1/2 of 224 +/- 66 h. A second infusion of the same dose of 131I-chimeric B72.3 was administered to four of these patients 8 wk after the first infusion. Two patients who had a high antibody response to initial infusion had an anamnestic antibody response, and the infused ch-B72.3 rapidly disappeared from the circulation with associated immune complexes and free 131I in the plasma. One patient with no initial antibody response had no antibody response and identical pharmacokinetics on second infusion. One patient with a modest transient antibody response to initial infusion had no antibody response on second infusion and a modest shortening of plasma circulation. Thus, the human immunoglobulin G4 isotype chimeric B72.3 monoclonal antibody has a plasma half-life 6 to 8 times as long as murine B72.3 and retains considerable immunogenicity in some patients which can adversely affect repetitive infusions.

Hairy cells possess more interferon receptors than other lymphoid cell types.

To investigate the possible direct effects of alpha-interferon (IFN-alpha) in hairy cell leukemia, IFN-alpha receptor expression by hairy cells (11 cases) was measured by a radiolabeling technique and compared with that of MOLT-4, chronic lymphocytic leukemia (15 cases), and various other leukemic and normal cell types. Purified peripheral blood and splenic hairy cells showed higher levels of receptor expression (approximately 1,000 +/- 200 binding sites/cell; 11 cases tested) than other normal and leukemic cell types. B cells from normal blood and tonsils showed low levels of receptors (approximately 120 +/- 100 binding sites/cell), while a range of B cell leukemias displayed intermediate levels of expression (100-500 sites/cell). In the 15 cases of chronic lymphocytic leukemia tested, 530 +/- 330 binding sites/cell) were demonstrated, the high standard deviation reflecting the fact that one third of cases had receptor levels comparable with those in hairy cell leukemia. Normal and hairy cell leukemia T cells, red cells, and platelets had no demonstrable IFN-receptors. These findings may be relevant to the efficacy of IFN in hairy cell leukemia.

The regulation of membrane-bound and secreted immunoglobulins in the human lymphoid cell line LICR-LON and human hybridomas.

The synthesis of membrane-bound and secreted immunoglobulin was investigated in the human line LICR-LON-HMy2, a cell line often used for the derivation of human hybridomas. PAGE-SDS analysis of immunoprecipitates obtained from 35S-methionine labelled cell lysates shows that LICR-LON synthesize a hitherto undetected membrane form of IgG (with a heavy chain of mol. wt 62,000) in addition to the secretor form of IgG already described (55,000 heavy chain). Tunicamycin treatment, pulse-chase experiments and Western blot analysis showed that both chains are synthesized as independent proteins. Hybridomas obtained after fusion of LICR-LON and human peripheral blood lymphocytes retained the ability of the parental cell line to synthesize gamma m and gamma s. Some of these hybrids synthesize and secrete IgM which presumably originates from the parental B-lymphocytes. Precipitation and PAGE-SDS analysis of membrane proteins after iodination of intact cells revealed only one heavy chain band, corresponding in size to that of the gamma m. No indication of the synthesis of the membrane form of IgM was found in the hybrids. These data show that the parental (lymphoid) phenotype (m and s-IgG) is codominant with the more differentiated phenotype (s-IgM) of the fusion partner cell (plasma cell). These observations are compatible with a class-specific m-s regulation operating on a different chromatin structure at the expressed Ig loci of each parental chromosome.

The mechanism of action of interferon-alpha (IFN-alpha) in hairy-cell leukaemia; Hu-IFN-alpha 2 receptor expression by hairy cells and other normal and leukaemic cell types.

To investigate the possible direct effects of interferon-alpha (IFN-alpha) in hairy-cell leukaemia, IFN-alpha receptor expression by hairy cells (HCs) (11 cases) was measured by a radiolabelling technique and compared with that of MOLT-4, chronic lymphocytic leukaemia (CLL; 14 cases) and various other leukaemic and normal cell types. Purified peripheral blood (PB) and splenic HCs showed higher levels of receptor expression (approx. 1000 +/- 200 binding sites/cell; 11 cases tested) than other normal and leukaemic cells types. Purified normal PB and tonsil B cells showed low levels of receptors (approx. 120 +/- 100 binding sites/cell), while a range of B-cell leukaemias displayed intermediate levels of expression (approx. 100-500 sites/cell). In the 15 cases of CLL tested, 530 +/- 330 binding sites/cell were demonstrated, the high standard deviation reflecting the fact that approximately one third of cases had receptor levels comparable with those in HCL. Normal and HCL T cells, red cells and platelets had no demonstrable IFN receptors. It is suggested that these findings may be relevant to the efficacy of IFN in hairy-cell leukaemia.

Immunoradiometric assay of human leukocyte interferon using monoclonal antibody.

Interferon research has been hampered by problems in its assay. The only widespread assays use tissue culture cells and compare some parameter of viral growth (such as viral RNA synthesis or host cell death) in the presence and absence of interferon. These complex biological assays, though sensitive, are laborious and subject to inherent variability. In particular, components other than interferon present in the assay sample often influence viral growth. There is clearly a need for a simple, direct, interferon assay; I now describe such an assay--an immunoradiometric assay utilizing monoclonal antibody.

Monoclonal antibodies by cell fusion.

Monoclonal antibodies produced by hybrid myelomas ('hybridomas') have become an important tool in those areas of research that use immunological techniques. In this article, David Secher describes some examples of monoclonal antibodies to illustrate some of the principles, problems and applications of the method.

Kinetics of internalisation and degradation of surface-bound interferon in human lymphoblastoid cells.

The binding of iodinated human interferon-alpha 2 (IFN-alpha 2) was studied on the human T cell line, Molt 4. After its initial binding to cells, the IFN is transferred to a trypsin-resistant compartment before appearing in the medium as TCA-soluble material, while the total cell-associated IFN declines to one-third of its maximum value after 3 h incubation. The Na+/H+ ionophore monensin did not prevent intracellular accumulation of IFN but did completely inhibit its breakdown. We interpret our results as evidence for receptor-mediated internalisation of IFN followed by intracellular breakdown.

From laboratory to clinic: the development of an immunological reagent.

Access to a wide range of high quality and increasingly sophisticated reagents and equipment has underpinned the great surge of knowledge in basic immunology and the growing interest in clinical immunointervention. In this article, the first in an occasional series on immunological research and development in industry, Sue Bright and colleagues outline the key steps in a development programme to take a humanized monoclonal antibody into the clinic. The procedures involved in developing such reagents, particularly for clinical use, are long and require considerable ingenuity and scientific creativity.

A monoclonal antibody for large-scale purification of human leukocyte interferon.

A clone of hybrid myelomas (NK2), secreting a mouse monoclonal antibody to human leukocyte interferon, has been isolated. The antibody neutralizes the antiviral activity of the interferon and, when covalently attached to a solid support and used as an immunoadsorbent, allows interferon purification of up to 5,000-fold in a single step.

Use of a monoclonal antibody specifically non-reactive with T cells to delineate lymphocyte subpopulations.

Rat monoclonal antibody M1/69.16 reacts with a heat stable antigen of mouse commonly expressed in the majority of cell types in blood, spleen, bone marrow and thymus, including cells of erythroid, myeloid and lymphoid series. However, subpopulations of cells in lymphoid tissues can be identified which are non-reactive with this antibody using the fluorescence-activated cell sorter. All surface Ig positive cells seem to react with M1/69.16 while more than 96% of Ig negative cells in spleen and lymph nodes are M1/69.16 negative. Most cells (80%-90%) in the M1/69.16 negative populations in spleen lymph nodes and bone marrow express Thy-l. Thus, peripheral T cells are specifically non-reactive with this antibody. In contrast, approximately 95% of thymocytes react with M1/69.16, leaving a minor population which is negative. The negative population (5%) is enriched in cells expressing high amounts of H-2 antigen and those bearing H9/25 antigen which is specific for lymphocyte subsets, indicating that M1/69.16 negative thymocytes represent a specific subpopulation, possibly "mature' thymocytes.

Effect of purified human interferon-alpha on the expression of differentiation antigens and mitogen reactivity of cultured human thymic cells.

Human thymic cells were cultured in vitro either alone or with the addition of a highly purified preparation of human interferon-alpha. Immunofluorescence techniques using a series of monoclonal antibodies showed that 2-day cultured thymocytes express a more mature phenotype (low HTA 1, high T3 and HLA-A,B,C) than normal, uncultured thymocytes. Interferon addition to the cultures results in a strong increment in the number of HLA+ cells and in the total amount of HLA expressed by the cultured cells. Experiments with purified cell populations showed that the cortical, immature, thymocyte was the target cell for interferon action. Phytohemagglutinin responses--but not interleukin 2 responses--were diminished after pretreatment of thymic cells with interferon. We suggest that interferon may favor a pathway of intrathymic differentiation phenotypically characterized by a high content of Class I HLA antigens.

Treatment of advanced condylomata acuminata with semi-purified and purified human leukocyte interferon.

Twelve patients with advanced condylomata acuminata were treated by systemic human leukocyte interferon (IFN) therapy. Semi-purified and purified preparations were both able to affect condylomatous growth. Treatment of the patients at various periods of their disease resulted in one complete remission, 6 partial remissions, 4 minimal responses while one case showed progressive disease. Side-effects were unexpectedly common in these advanced patients and 4 of them had to stop IFN treatment.

Monoclonal xenogeneic antibodies to murine cell surface antigens: identification of novel leukocyte differentiation antigens.

Hybrid myeloma cell lines secreting monoclonal antibodies to mouse cell surface antigens have been prepared. Spleen cells from a DA rat immunized with B10 mouse spleen cells that had been enriched for T cells were fused to cells from a nonsecreting mouse myeloma line (NSI). The presence in the culture supernatants of antibodies binding to mouse spleen cells was tested by a binding assay with 125I-labeled anti-rat IgG. From a large number of positive cultures, ten independent hybrid clones were purified, each secreting a different antibody. Each antigenic target was analyzed by (a) gel electrophoresis of immunoprecipitated 125 I-labeled cell surface molecules, (b) heat stability, (c) strain and species distribution and (d) cross-inhibition of binding of different monoclonal antibodies. It was concluded that the ten monoclonal antibodies regognized four types of antigen. One was the heterophile, heat-stable, Forssman antigen. The second (mol.wt. 210 000) appears to be a major 125I-labeled lymphoid cell surface protein. The third, a minor component of spleen cells, was precipitated as two polypeptides of mol.wt. 190 000 and 105 000. Five IgG-secreting clones identify the fourth antigen, a heat-stable, possibly glycolipid component expressed on mouse red blood cells and also on thymocytes. Cross-inhibition studies suggest that these last monoclonal antibodies bind to overlapping, but not identical, determinants. The class and chain composition of the monoclonal antibodies were studied by gel electrophoresis, isoelectric focusing and ability to lyse red blood cells and thymocytes.

Monoclonal antibodies and cell surface antigens.

Antibody chains are encoded in three gene clusters containing genes for the variable and constant regions. V and C genes are separated in germ line and during differentiation a rearrangement takes place. But even after this rearrangement the V and C coding sequences are not contiguous. A final splicing must take place in committed cells between the transcription of a discontinuous V-and C-region DNA and the expression of a continuous mRNA coding for an antibody chain. Analysis by cell fusion indicates that the splicing is cis. When two antibody-producing cell lines are fused, the resulting hybrids express the two antibodies that characterize the parental lines. Permanent cell lines producing antibody of predefined specificity have now been derived in this way. Spleen cells from hyperimmunized donors are fused with myeloma cells and a proportion of the hybrids that are established synthesize and secrete antibodies directed against the immunogen. The heterogeneous cell population can be cloned and propagated. This is a potent way of producing monospecific antibodies to complex antigens such as cell membranes and transplantation antigens. Monoclonal xenogeneic antibodies to rat cell-surface membranes have proved very valuable for characterizing and separating rat lymphocyte subpopulations. In more recent experiments, monoclonal xenogeneic antibodies to mouse and human cell-surface antigens have also been produced which permit the characterization of the hitherto undescribed differentiation antigens.