Cell surface antigens of L-1210 leukemia recognized by monoclonal antibodies

Conventional antisera to L-1210 leukemia were being prepared in our laboratory for nearly a decade and consistently the only specificity detectable was anti Mammary Leukemia antigen (ML). Serological analysis of five monoclonal antibodies obtained following the same immunization schedule showed more diverse pattern of reactivity. Two antigens detected belong to oncofetal category. The third one is differentiation antigen Ly-6 and the nature of two others, expressed on leukemic cells only, remains at present unclear. Thus none of the clones analysed produces antibodies to ML antigen. Our previous analysis of cell surface antigens of L-1210 leukemia with the use of conventional antisera has already been described. This paper presents the results of applying monoclonal antibodies in a comparable studies.     

Analysis of surface antigen expression per cell of human osteogenic sarcoma cells by fluorescence-labelled monoclonal antibodies

The relationship between total surface antigen expression per cell (means) - measured by fluorescence-labelled monoclonal antibodies (fluorescence-histograms) and the distribution of cells in the cell cycle (DNA-histograms) and size-scattergrams (cell sorter FACS-IV) were analysed in drug treated unsynchronized and synchronized osteogenic sarcoma cells (2OS) in vitro. Drugs with various sites of action in the cell cycle were used. Adriamycin, Vindesine, in concentrations applied accumulate cells in G2 + M phase. Methotrexate arrests cells in the boundary of G1/S phase. Size-scattergram and DNA-histogram analysis have shown that the entrance of cells to the cell cycle is usually accompanied by an increase in the cells size and amount of their DNA. The size of the cells influenced antigenic expression much more than the distribution of the cells in the various cell cycle phases: in the bigger cells the expression per cell was more pronounced. The increase of antigen expression was the highest for Adriamycin and for Methotrexate treated cells. However, this increase was limited and never exceeded plus 50% in relation to the control. This relatively low difference resulted from the fact, that a given phase of the cell cycle included cells markedly heterogenic in respect of size and antigenic content. It was also shown that lower concentration of serum in culture medium and confluent growth of older cultures decrease surface antigen expression per cell.     

Production of monoclonal antibodies against hepatitis B surface antigen (HBsAg) by somatic cell hybrids

Hybridomas secreting anti-HBs were produced by fusion of either adw or ayw HBsAg primed mouse spleen cells with either P3 X63 Ag8 or P3 NSI 1 Ag4 1 mouse myeloma cell lines. Individual anti-HBs secreting clones were isolated by limiting dilution procedures, and six cell lines have been established, namely, BX182, BX259, BX248, CN324, DN283, and DN296. Progenies of each cell line were derived from a single clone obtained from three subclonings of six anti-HBs positive initial fusion colonies. Clones were passaged in tissue culture and as tumors in syngeneic mice for upwards of six months. Anti-HBs of each line showed characteristic reactivity (detection) patterns in radio-immunoassay using different antigen subtype solid phases followed by either ¹²⁵I-HBsAg or ¹²⁵I-goat anti-mouse IgG probe. The specificity of the anti-HBs from each clone for the subdeterminants of HBsAg was identified by their reaction with ¹²⁵I-HBsAg ligands of several subtypes in a radioimmunoprecipitation assay. Four types of reaction were identified and correlated to the conventional serological subtyping definitions; they were anti-HBs/a (BX259 and CN324), anti-HBs/d (BX182), and possibly anti-HBs/w (BX248 and DN296) and anti-HBs/y (DN283). These monoclonal antibodies will be important for the elucidation of the antigenic structure of native HBsAg and will provide valuable reagents for both antigen detection and subtyping.     

Human monoclonal antibodies reactive with cell surface antigens on human leukemia cell lines: many antibodies are (auto)antibodies

Primary Epstein Barr Virus (EBV) transformants from peripheral blood mononuclear cells (PBM) established in macrocultures were screened for the secretion of antibodies reactive with cell surface antigens on one or another of two indicator human leukemic cell lines and fused with the HMMA2.11TG/O human fusion partner. Human monoclonal antibodies (HuMAbs) were readily obtained. Relative oligoclonality of the primary EBV macrocultures was documented by the number of antibody secreting hybridomas (1-100%). The method permitted preselection for fusion of transformants producing antibodies of certain specificities and/or class. Fourteen HuMAbs, primarily of the IgM class, have been obtained. Those IgM HuMAbs obtained from patients with active diseases, e.g. Acute Lymphoblastic Leukemia (3 HuMAbs), and HIV infection (4 HuMAbs), were found to have a relatively broad spectrum of reactivity with cell lines of various hematopoietic lineages and normal cells, although several show selective reactivity with T cell lineage tumors or a selected population of cells. HuMAbs from normal donors of both the IgG and IgM class were obtained. The IgM HuMAbs from one volunteer reacted primarily with autologous and allogeneic macrophages (autologous PBM from the other patients were not available) as well as a diverse number of hematopoietic cell lines. From others, the IgG HuMAbs demonstrated a more restricted spectrum of reactivity, while the IgM HuMAbs reacted with both autologous and allogeneic normal cells. Thus, the B cell repertoire contains cells capable of secreting cell surface reactive antibodies and many of these antibodies express characteristics of autoantibodies. Those that did not react with autologous or allogeneic PBM may react with other autoantigens which have been expressed on the malignant cells used as screening targets or may represent true antitumor antibodies.     

Localization of hepatitis B surface antigen epitopes present on variants and specifically recognised by anti-hepatitis B surface antigen monoclonal antibodies

Small hepatitis B surface antigen (HBsAg) is considered to be the best marker for the diagnosis of Hepatitis B virus infection. However, HBsAg variants with mutations within the "a" determinant may be poorly or not detected by diagnostic assays. Three anti-HBsAg monoclonal antibodies (6H6B6, 27E7F10, and 2G2G10), directed against conformational epitopes, were tested for their ability to detect the wild-type HBsAg as well as variant forms and their respective epitopes were localised on the HBsAg sequence by using the phage-displayed peptide library technology. Whereas 6H6B6 did not detect mutations T123N, S143L, D144A and G145R, 27E7F10 binding was affected by mutations P120T and G145R. In contrast, 2G2G10 reacted strongly with all tested variants including variant with the G145R mutation. Part of the 6H6B6 epitope was located in the major hydrophilic region (MHR) at residues 101-105, the 27E7F10 epitope (residues 214-219) was located near the C-terminal end of the antigen and the 2G2G10 epitope at residues 199-208, within the theoretical fourth transmembrane helix. The 2G2G10 epitope localisation brings information about the HBsAg structure and the validity of established topological models. Finally, 2G2G10 is a valuable tool for HBsAg variant detection that is used as capture phase in a new bioMérieux diagnostic assay, which is currently in development.     

High affinity human monoclonal antibodies directed against hepatitis B surface antigen

Peripheral blood mononuclear cells from donors immunized with hepatitis B vaccine (Pasteur Hevac B) were transformed with Epstein-Barr virus. Two polyclonal cell lines, producing antibodies to hepatitis B surface antigen were established and cloned. Seven clones were isolated; they secreted between 10 and 20 micrograms/ml of HBs specific IgG1 kappa or lambda antibody with anti-HBs titer of 300-800 IU/ml. These human antibodies expressed the anti 'a' specificities and had high affinity and avidity; their potential use as reagents for hepatitis B virus detection and for passive immunotherapy is under study.     

Quantitative analysis of cell surface HLA structures by means of monoclonal antibodies

Quantitative data on the binding of murine monoclonal antibodies to whole human lymphoblastoid lines and peripheral blood lymphocytes (PBL) are reported. Antibodies reacting with β₂m or a common part of the HLA heavy chains and nonpolymorphic determination of the DR dimer were used. The equilibrium constant (K) of the reaction and the total number of antigenic determinants was graphically estimated. For the above-mentioned antibodies. K ranged between 5 × 10⁸ and 4 × 10⁹ I lmole at 0°C and progressively decreased with the increasing temperature. T cells expressed less HLA and β₂m determinants that the B cells. The number of determinants per surface unit is higher on the B cell from PBL than on E.B. virus-transformed cell lines and is generally very low, suggesting that the complement-dependent cytotoxic activity is a phenomenon depending on membrane fluidity. A portion of β₂mseems not to be bound to the HLA heavy chains on B cells as well as on T line surface, as already shown for Molt 4 line.     

The GIX antigen of murine leukemia virus: an analysis with monoclonal antibodies

The G_IX antigen of murine retrovirus gp70 is a marker for virus replication in various cell types, as well as for T-cell differentiation and leukemogenesis in particular mouse strains. The serological definition of G_IX depends on the cytotoxicity of a particular rat antiserum for thymocytes from strain 129 mice. We have found that two rat monoclonal antibodies, elicited by immunization with AKR ecotropic virus or AKR leukemia cells, co-type closely with G_IX in several assay systems. Like G_IX, the epitopes identified by the monoclonal antibodies are amplified on thymocytes during leukemogenesis in AKR mice. These antibodies are cytotoxic for G_IX⁺ leukemia cells and for fibroblasts infected with G_IX⁺ viral serotypes. Though absorbed by G_IX⁺ thymocytes of various strains, the antibodies are not cytotoxic for these cells. We ascribe the latter result to lower representation of antigenic sites on normal thymocytes, and postulate that more than one epitope may participate in classical G_IX-mediated cytotoxicity. The monoclonal antibodies do not react with viral env products synthesized in the presence of the inhibitor of glycosylation, tunicamycin. Reactivity with the antibodies appears to require a stable configurational change in the env precursor protein coinciding with glycosylation, rather than direct participation of carbohydrate in the antigenic site. Thus subsequent enzymatic removal of carbohydrate chains in vitro does not alter reactivity with the antibodies.     

In vitro neutralization of hepatitis B virus by monoclonal antibodies against the viral surface antigen

In vitro HBV infection and neutralization were assayed using an anti-preS1 murine monoclonal antibody (1B3) and anti-preS2 (H69K) and anti-S (CS131A) murine-human chimeric antibodies. The 1B3 (lgG1) and H69K (lgG1) was constructed previously and the CS131A was constructed for this study by expressing stably the chimeric heavy and light chains in Chinese hamster ovary cells and purifying from the culture supernatant. Previous study showed that the H69K and CS131A recognize known virus-neutralizing epitopes, while the 1B3 does not. For the assays, adult human hepatocyte primary culture was infected with the adr or ayw subtype of HBV, and the infectivity and subsequent replication was confirmed both by measuring the kinetics of HBsAg secretion by the infected cells and detecting the intermediate replicative form of HBV DNA in the cells. Next, the hepatocytes were infected with the adr or ayw subtype of the virus that had been preincubated with various concentrations of each of the antibodies and the neutralization of HBV was analyzed. The results showed that the anti-preS2 and anti-S chimeric antibodies exhibited neutralizing activity against both the adr and ayw subtypes of the virus, with approximately 1,000 and 2,000 times higher specific activity than polyclonal hepatitis B immune globulin, respectively, but the anti-preS1 antibody scarcely neutralized the infection. The neutralizing activities of the antibodies were consistent with their epitope specificity and antigen-binding affinity, suggesting that this neutralization assay is specific. The in vitro neutralization assay will be useful for evaluating the neutralizing activity of anti-HBV antibodies before in vivo testing in chimpanzees. J. Med. Virol. 52:226–233, 1997. © 1997 Wiley-Liss, Inc.     

Two screening methods show different antigen recognition patterns for four monoclonal antibodies to Candida albicans cell surface

Four murine monoclonal antibodies (mAbs) showing similar reactivity with a cell-wall extract and mannan preparation obtained from Candida albicans were examined for epitope specificity. An enzyme-linked immunosorbent assay (ELISA) was used to determine total mAb binding to extracted cell-wall antigen when each mAb was reacted alone or in competition with a second mAb. This analysis suggested that three mAbs recognized the same determinant, which differed from that recognized by the fourth. The reactivity of these mAbs was also examined by indirect immunofluorescence assay with both yeast and germ tube forms of the dimorphic fungus. The three mAbs assigned the same epitope-specificity by ELISA showed two different patterns of reactivity with immunofluorescence. This discrepancy is discussed with respect to the postulated structure of mannan and the method of analysis.     

Structural characterization of viral neutralizing monoclonal antibodies to hepatitis B surface antigen

In this study we describe the viral neutralizing activity of murine monoclonal antibodies (MAb) specific for hepatitis B surface antigen (HBsAg). This viral neutralizing activity was assessed in vitro by employing Hepatitis Delta Virus (HDV) and human hepatocytes as target cells. To further characterize these viral neutralizing antibodies we generated a panel of anti-idiotypic (anti-Id) reagents and serologically characterized these antibodies for epitope specificity, Id specificity, and Id heterogeneity. Direct binding and competitive inhibition solid phase enzyme immunoassay have demonstrated that two murine MAb specific for HBsAg (anti-HBs), designated A1.2 and A3.1, recognize similar or overlapping epitopes on HBsAg, while monoclonal anti-HBs, designated A2.1 recognizes a unique HBsAg epitope. Further, Id analysis using monoclonal and polyclonal anti-Id reagents have identified both a private and a cross-reactive Id, respectively, on the anti-HBs, A1.2 preparation. The source of the idiotypic cross-reactivity between A1.2 and A3.1 has been identified, using Western blot analysis, to conformational determinants expressed by the heavy (H) and light (L) chains of these monoclonal anti-HBs. Lastly, the intrastrain antibody repertoire induced following HBsAg immunization was found to be relatively restricted in heterogeneity by clonotype analysis using isoelectric focusing and affinity immunoblot analysis. Interspecies variability in the anti-HBs response was observed based on epitope recognition using purified anti-HBs from a variety of species.     

Rabbit leukocyte surface antigens defined by monoclonal antibodies

Several monoclonal antibodies (mAb) against rabbit leukocytes were characterized in binding and functional studies. mAb 1.24 stains thymocytes, bone marrow cells, peripheral T and B cells and blood monocytes. T cells express more 1.24 antigen than B cells. In the absence of added complement (C), mAb 1.24 inhibits alloantigen-, concanavalin A (Con A)-, and phytohemagglutinin (PHA)-, but not pokeweed mitogen (PWM)- or anti-immunoglobulin (Ig)-induced cell proliferation. It also strongly blocks anti-sheep erythrocyte plaque-forming cell responses. A second mAb, designated 4.B9, binds to 20% of thymocytes and to most, if not all, peripheral T cells and in vitro-activated T cell blasts. A third one, 10.B3, is reactive with the nearly entire thymocyte and a major peripheral T cell population. Two-color membrane immunofluorescence reveals the presence of a small population of peripheral blood leukocytes which bear surface Ig and are weakly stained by mAb 4.B9 and 10.B3. Without C, both 4.B9 and 10.B3 inhibit Con A- and PHA-induced mitogenesis, but have no effect on PWM-, antigen-, or alloantigen-induced cell proliferation. Depletion of 4.B9+ cells by panning or complement lysis completely abrogates proliferative responsiveness to antigen and alloantigen, significantly reduces responsiveness to the T cell mitogens Con A and PHA, but enhances that to the B cell mitogen anti-Ig. A fourth mAb, 12.C7, binds to 60% of thymocytes and to 10-30% of peripheral T lymphocytes at high-level fluorescence. T cell blasts obtained in mixed leukocyte reactions are partially stained by mAb 12.C7, while those obtained after Con A or PHA activation are not. In addition, mAb 12.C7 is completely unreactive with B cells or monocytes. Without complement, it does not seem to interfere with any of the in vitro functions tested. All antigens studied here do not appear to be expressed in nonleukon tissues, as they do not bind to erythrocytes and are absent from brain, heart, liver and kidney as shown by quantitative absorption analysis.     

Detection of islet cell specificity of monoclonal islet cell surface antibodies by means of double-staining immunofluorescence

We have generated monoclonal antibodies (mcab) reactive with islet cell surface antigens. 10 different mcab were characterized regarding their islet cell binding specificity by means of a modified double immunofluorescence test. At this assay, the monoclonal islet cell surface antibodies were visualized on rat islet cells by indirect immunofluorescence with fluorescein isothiocyanate-labelled antibodies against mouse immunoglobulin. The alpha and beta cell specificity was determined by indirect immunofluorescence using anti-glucagon or anti-insulin serum and a tetramethyl rhodamine isothiocyanate-labelled second antibody. The target islet cell suspension used contained 61% beta and 23% alpha cells. The monoclonal antibody K28D6 preferentially reacted with alpha cells. The binding of K29aC6 and K56aF3 indicates a high specificity against beta cells. The remaining 7 antibodies were reactive with alpha as well as with beta cells.     

Recognition of several idiotopes on a monoclonal anti-hepatitis B virus surface antigen using monoclonal anti-idiotypic antibodies.

A monoclonal murine antibody H3F5 directed to the a determinant of hepatitis B surface antigen (HBsAg) was used to raise several monoclonal anti-idiotypes. Cross-blocking experiments between the anti-idiotypes and the patterns of inhibition produced by a number of other monoclonal anti-HBsAg, generated in the same fusion as H3F5, identified three idiotopes on H3F5 which were shared to varying degrees with the other anti-HBsAg monoclonals. One behaved as a dominant cross-reacting idiotope (CRI) in that it appeared strongly in 5/7 monoclonal idiotypes. The CRI could represent an important target for regulation by anti-idiotope. Monoclonal antibodies have many advantages over polyclonal sera in the detailed analysis of idiotope structures.     

Characterization of mouse monoclonal antibodies specific for Friend murine leukemia virus-induced erythroleukemia cells: friend-specific and FMR-specific antigens

Monoclonal antibodies were derived from hybrid cell lines produced by fusing mouse myeloma cells with spleen cells from mice recovering from Friend virus-induced erythroleukemia. Of the 17 clones characterized, two appeared to have the Friend, Moloney, Rauscher pattern of specificity. One of these was specific for the envelope protein, gp70, and the other reacted with a core protein, p15. Seven other anti-gp70 clones and one anti-p15 clone were restricted in reactivity to cells infected with Friend or Rauscher viruses only. One clone reacted with p15E and recognized this protein on many ecotropic and dual-tropic viruses. In addition, six IgM antibodies were obtained which appeared to recognize nonviral antigens present only on leukemia cells of the erythroid lineage. Six monoclonal antibodies of complement-fixing immunoglobulin classes with specificity for gp70 or p15 were compared for their ability to bind or lyse erythroleukemia cells in the presence of complement. Individual antibodies to the same viral protein appeared to differ markedly in their ability to mediate cytolysis.     

A rapid one-step radiometric assay for hepatitis B surface antigen utilising monoclonal antibodies

A two-site antigen assay for HBsAg has been developed that employs 3 monoclonal antibodies. The antibodies were selected for their high affinity and their particular epitope specificity to establish an assay with a sensitivity for the antigen comparable with that of a conventional assay with heterologous antisera. In addition, by selecting a monoclonal antibody for use as a tracer which does not compete for antigenic binding sites with the solid-phase monoclonal antibodies, it has been possible to perform a two-site assay in a single 1 h incubation step, achieving the same degree of sensitivity. This principle of using monoclonal antibodies in a one-step assay therefore gives advantages of speed and simplicity over assays using heterologous antisera and would be applicable to a variety of antigen assays for which appropriate monoclonal antibodies are available.     

Importance of antigen specificity for complement-mediated lysis by monoclonal antibodies

Lysis of human lymphocytes by autologous complement had been studied using a range of monoclonal antibodies against different antigens. Antigen specificity (and not antibody isotype) was the most important factor which influenced cell lysis and this could not be accounted for merely by differences in surface density between antigens. Three antigens with comparable surface density were studied in detail: CAMPATH-1 (lytic), major histocompatibility complex class I (lytic) and leukocyte common antigen (poorly lytic). C1q binding was roughly proportional to antibody binding and dependent on antibody isotype. However, the lytic antibodies were much better able to bind and activate whole C1 than the poorly lytic ones. This result would not have been predicted from traditional concepts of complement activation but can be interpreted in the light of models for C1 activation which involve Fc-Fc interactions, Fc-C1r2s2 interactions and a critical C1q stem-arm angle for C1 binding and activation.     

Differentiation antigen on murine B lymphocytes defined by monoclonal antibodies

Spleen cells from an AKR/J X DBA/2J F1 mouse immunized with NZB/BIN spleen cells were fused with SP2/0-Ag14. Two hybrid cell lines, B220-1 and B220-2, were established that secreted antibody to the B-lineage specific B220 antigen. B220-1 and B220-2 are present on 45-55% of splenic and bone marrow lymphocytes and absent from thymus. By flow cytometry, all immunoglobulin-bearing cells were stained by these monoclonal antibodies. Although these monoclonals do not stain thymocytes, they do react weakly with Lyt-2+ peripheral T cells. Dual parameter analysis of B lymphocytes using RA3-3A1 or 14.8 show that these monoclonals recognized the same population. Prior incubation with RA3-3A1 or 14.8 was unable to completely block the binding of B220-1 or B220-2, implying that the epitopes recognized are different from the previously described monoclonal antibodies. Immunoprecipitation of the splenic lymphocyte reveals a molecule which migrates on SDS-PAGE as a single band with MW of 220,000 daltons. Expression of the distinct antigens recognized by B220-1 and B220-2 varied among mouse strains, indicating previously unappreciated polymorphism of the B220 molecule. These monoclonals are useful for cytotoxic elimination of B cells and for three-color flow cytometry.     

T cell activation by monoclonal antibodies bound to tumor cells by a cell surface displayed single-chain antibody.

Tumor cells often lack the costimulatory molecules necessary for T cell activation. However, the transformation of cells with more than one stimulatory molecule is a difficult procedure. We therefore developed a retroviral vector for the expression of a cell membrane anchored single-chain antibody fragment (scFv) directed against the hapten 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one (phOx). Proteins and peptides can be readily modified with this hapten, thus, enabling them to be bound to cells with the cell surface displayed anti-phOx scFv. To test combinations of surface-bound stimulatory molecules on T cell activation, SK-Mel63 human melanoma cells expressing the membrane anchored anti-phOx scFv were incubated with phOx-labeled mAbs against CD3, CD28 and CD5. Cells presenting a given mixture of modified anti-CD3 and anti-CD28 molecules stimulated T cell activation better than any single antibody and a given mixture of anti-CD3, anti-CD28 and anti-CD5 provided a stimulatory response higher than the best double combination. However, the relative concentrations are very important and must be carefully chosen. Concentrations of antibodies giving good T cell responses when used alone can block synergistic effects.