Generation of effector T cells expressing chimeric T cell receptor with antibody type-specificity.
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Animals↗
Biomedical subjects
Z Eshhar
Publications and source records attributed to Z Eshhar.
Anti-IgE monoclonal antibodies directed at the Fc epsilon receptor binding site.
In a search for the region in the IgE molecule, which is recognized by the Fc epsilon receptor (Fc epsilon R) on mast cells and basophils, we have generated and characterized anti-IgE monoclonal antibodies (MAbs). The novel rat anti-mouse IgE MAb described herein (denoted 84-1c) interacts with an antigenic determinant which is associated with the Fc epsilon R recognition site on the IgE molecule. The MAb can bind to the Fc epsilon of IgE and block its binding to rat basophil leukemia (RBL) cells. The epitope recognized by 84-1c MAb was completely masked by the Fc epsilon R either in its cellular or soluble form. This epitope was dependent on the native conformation of the IgE molecule and differed from the ones that were recognized by the anti-IgE MAbs we described before.
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Monoclonal antibodies specific to the alpha-subunit of the mast cell's Fc epsilon R block IgE binding and trigger histamine release.
In an attempt to block the interactions between IgE and its receptor on mast cells (Fc epsilon R), we have established anti-Fc epsilon R monoclonal antibodies (mAb) by fusion of myeloma cells with mouse splenocytes immunized with irradiated rat basophilic leukemia (RBL) cells. Two anti-Fc epsilon R mAb were obtained (denoted 4.7 and 5.14) that could specifically bind to RBL and mast cells. This binding could be inhibited by IgE. The mAb and their F(ab')2 fragments inhibited 125I-IgE binding to RBL cell and triggered cell degranulation. The Fab' fragments, on the other hand, could only inhibit IgE binding but did not stimulate cell degranulation. Furthermore, these monovalent fragments inhibited RBL and mast cell degranulation induced by IgE-antigen complexes both in vitro and in vivo in the passive cutaneous anaphylaxis reaction. The number of mAb 4.7 and 5.14 molecules bound per RBL cells was similar to that of IgE; nevertheless, mAb 4.7 and 5.14 recognized different epitopes on the IgE receptor. Immunoprecipitation and immunoblotting analysis demonstrated that the mAb reacted with the alpha-subunit of the Fc epsilon R. Our findings establish the anti-Fc epsilon R mAb as a useful reagent for the isolation and characterization of the Fc epsilon R's alpha-subunit and the monomeric (Fab') for blocking the IgE-Fc epsilon R interactions.
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Anti-anti-IgE idiotypic antibodies mimic IgE in their binding to the Fc epsilon receptor.
The binding site of some anti-idiotypic antibodies (anti-Id) can appear as a structural image of the antigen and as such may mimic its biologic activity. We raised anti-anti-IgE antibodies in an attempt to obtain anti-Id capable of interacting with the Fc epsilon receptor (Fc epsilon R). Guinea pigs were immunized with purified murine monoclonal antibodies (mAb) that had been found to react with epitopes closely related to the site on the IgE molecule which is recognized by the Fc epsilon R. After only two injections, we could detect in the immune sera anti-Id that inhibited the binding of IgE to the anti-IgE mAb used as immunogens. However, only after 10 immunizations over a period of about 6 months could we detect antibodies that competed efficiently with the binding of IgE to rat basophilic leukemia (RBL) cells. The "IgE-like" anti-Id could be affinity purified from immunosorbents made of the anti-IgE mAb. F(ab')2 and Fab' fragments were as effective inhibitors of IgE binding as the intact anti-anti-Id antibodies. Some of the anti-Id caused RBL degranulation and all of them, like IgE, inhibited the binding of specific anti-Fc epsilon R mAb to RBL cells. In summary, by hyperimmunization with anti-IgE mAb we could obtain anti-Id whose antigen-binding site is recognized by the mast cell receptor specific to the Fc portion of IgE.
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RAB-1: a new monoclonal antibody to leukemic hairy cells.
RAB-1, a new monoclonal antibody (McAb) to human leukemic hairy cell (HC) was produced. Using indirect immunofluorescence methods and microscopic or flow cytometric analysis, it was found that the RAB-1 antigen was expressed on few resting B cells and not on resting T lymphocytes, platelets, monocytes, erythroid and myeloid cells. RAB-1 expression on malignant cells was as follows: strongly positive in 15/15 hairy cell leukemia (HCL), negative with non-T and T-acute lymphoblastic leukemia and T-chronic lymphocytic leukemia (CLL); weakly expressed on myeloma and Waldenstrom cells; moderately on 10-25% of the cells in 4/10 B-CLL and 6/10 B lymphomas and in 7/7 B-prolymphocytic leukemia (PLL). Amongst human cell lines that were tested, RAB-1 reacted strongly with one HC line, moderately with the EBV-lymphoblastoid Daudi and Raji Burkitt's lines and was not expressed on Ramos, ALL, myeloid and erythroid cell lines. Normal B cells activated with PWM or anti-mu beads, and malignant B cells activated with anti-mu and TPA did not show an increase of expression of RAB-1 antigen. Interestingly, 30-40% of T4-Class II antigen positive cloned cells and T cells activated with PHA and Con.A expressed RAB-1, suggesting that this McAb recognizes surface molecule, newly induced during T-cell activation and constitutively expressed on HC and some B-cell malignancies.
Antibodies, Monoclonal↗
T cell derived IgE binding factors inhibit IgE-specific rosettes on basophilic leukaemic cells and lymphocytes.
T cell derived IgE-binding factors inhibited the formation of IgE-specific rosettes on both rat basophilic leukaemic cells and lymphocytes. This was demonstrated by IgE-binding factors released from either IgE-pulsed T cells or adherent cells as well as from IgE-binding factor producing hybridomas. The different binding factors inhibited the IgE-rosettes to a different degree when tested on basophils and lymphocytes, most likely due to the higher affinity for IgE of the former cell type. The results indicate that rat basophils and mouse T cells may recognize common epitopes on the Fc portion of IgE.
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A human monoclonal antibody to insulin.
To elucidate the immune aspects of insulin-dependent diabetes mellitus (IDDM), we attempted to generate human monoclonal anti-insulin antibodies by fusing peripheral blood lymphocytes obtained from 10 insulin-treated IDDM patients with cells from a human lymphoblastoid cell line. Hybridomas that secreted immunoglobulins appeared in 9 of 400 wells. One of these hybridomas secreted anti-insulin antibody of the IgM class. The lymphocytic partner of this hybridoma was obtained from an IDDM patient who had undetectable levels of antibodies to insulin in his serum. Thus, by employing the hybridoma technique, it was possible to reveal the presence of insulin-sensitized B-lymphocytes in a patient who was serologically negative for anti-insulin antibodies. The monoclonal antibody recognized intact human insulin and insulins of other species, but not isolated A- and B-chains. This indicates that the antibody was functionally an autoantibody directed to an epitope formed by the native conformation of a highly conserved portion of the insulin molecule. This is the first report of a human hybridoma antibody to insulin.
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Relationships between epitopes on IgE recognized by defined monoclonal antibodies and by the FC epsilon receptor on basophils.
The present study investigated whether the sites on the FC region of the IgE molecule, recognized by different anti-IgE monoclonal antibodies (mAb), are identical to those recognized by the Fc receptor (Fc epsilon R). The anti-IgE mAb recognize different clusters of epitopes on the Fc region of IgE and could interfere to different degrees with the binding of IgE to mast cells and basophils, but still recognized cell-bound IgE. Analysis of the stoichiometry and affinity binding of 125I anti-IgE mAb Fab' to free IgE have revealed that anti-IgE mAb of one group (51.3) recognized three repetitive determinants on the IgE Fc portion, and another group (95.3) recognized only one determinant. When these stoichiometric studies were performed with cell-bound IgE, it was found that only one of the sites recognized by 51.3 mAb was involved in the Fc epsilon R binding site. On the other hand, the site recognized by 95.3 mAb was not the Fc epsilon R binding site. Such findings establish mAb 51.3 as a useful tool for isolating the IgE peptides involved in the binding site to the receptor.
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Organ specific neoantigens reactive in the leukocyte adherence inhibition assay: affinity purification of human colon carcinoma antigen and its cross-reactive protein using monoclonal antibodies.
A monoclonal antibody (mAb) was prepared against a semipurified preparation of an organ specific neoantigen (OSN) reactive in the leukocyte adherence inhibition (LAI) assay. The mAb (LC20.1) induces a positive LAI response when incubated with leukocytes of normal individuals in the presence of OSN derived from either human colon or lung carcinoma cell lines. Absorption of crude OSN preparations from these cell lines on immobilized LC20.1 mAb eliminates all the LAI reactive material suggesting that the mAb recognizes a common determinant on OSN from both colon and lung carcinomas. The LC20 mAb was used to affinity purify the colon cancer OSN as well as a cross-reactive normal protein from the urine of colon cancer patients and healthy donors, respectively. The colon cancer OSN and normal cross-reactive protein display an apparent molecular weight of 29,000, have a similar linear tryptic peptide map, and are indistinguishable by isoelectric focusing analysis. Regardless of the molecular similarity, only the colon cancer OSN preparation could induce a positive LAI when incubated with leukocytes of colon cancer patients. Seven additional anti-colon cancer OSN mAbs were prepared against purified material. These mAbs can be divided into three groups, each of which recognizes a distinct antigenic determinant that is shared by the colon cancer neoantigen and its cross-reactive normal protein.
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Induction of Fc epsilon receptors on normal murine T cells and IgE binding factor(s) by cross-linked IgE or IgE-pulsed adherent cells.
This study aimed to compare the efficiency and extent of induction by monomeric versus cross-linked IgE of specific receptors for IgE on normal murine splenic T cells (Fc epsilon R-T), and to study the ability of IgE-pulsed splenic adherent cells to induce receptors for IgE on T cells. Chemically cross-linked IgE was found to be both more effective and more efficient than monomeric IgE in inducing Fc epsilon R-T as measured by the ability of IgE-pulsed T cells to form specific rosettes with IgE-sensitized trinitrophenylated sheep red blood cells (TNP-SRBC). This phenomenon was dependent on both DNA and protein synthesis, suggesting that induction caused the production of new IgE receptors. It was also found that cross-linked but not monomeric IgE-pulsed normal murine adherent cells as well as their cell-free products could actively induce significant levels of specific Fc epsilon R-T. Both cross-linked IgE-pulsed T cells and adherent cells released IgE binding factor(s). These materials were capable of specifically inhibiting the binding of IgE to rat basophilic leukaemic cells (RBL) in vitro and to rat tissue mast cells in vivo. Collectively, these data provide further evidence to suggest that polymerized forms of IgE and adherent cells play important roles in the regulation of IgE responses.
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Use of monoclonal antibodies to a human cytotoxin for its isolation and for examining the self-induction of resistance to this protein.
Crude preparations of cytotoxins (CTXs) produced by human peripheral blood mononuclear cells exert a marked cytotoxic effect when applied to cells in the presence of cycloheximide but in its absence can induce resistance to cytotoxicity. To examine the relationship between these cytotoxic and protective activities, we attempted to fully dissociate the CTX from the other proteins secreted by mononuclear cells. Mice injected with preparations of the cytokines secreted by peripheral blood mononuclear cells developed significant titers of serum antibodies to CTX(s). Splenocytes of such immunized mice were fused with NSO myeloma cells; a few among the resulting hybridoma cells secreted CTX-binding antibodies. Immunoadsorbents constructed with a monoclonal antibody produced by one of these hybridomas were used to purify to homogeneity a CTX (Mr approximately 17,500) from crude preparations of cytokines, by a single adsorption and elution cycle. Purified CTX was cytotoxic in the presence of cycloheximide but in its absence induced resistance to cytotoxicity; this resistance was manifested by decreased vulnerability to CTX in a subsequent incubation in the presence of cycloheximide. We conclude that CTX itself can induce certain changes in cells, which are reflected in resistance to its own cytotoxic effect.
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Protective monoclonal antibody against Schistosoma mansoni: antigen isolation, characterization, and suitability for active immunization.
Monoclonal antibodies that bind to the surface of developing schistosomula were generated from the spleens of chronically infected mice that were boosted with cercarial glycoproteins. The two most reactive monoclonal antibodies, denoted 152-66-9B and 152-66-1C, were used for identification of surface antigens. The antigen detected by these monoclonal antibodies persisted on the surface of the developing larva for 72 hr posttransformation. This monoclonal antibody effected complement-mediated killing of schistosomula in vitro as efficiently as infected mouse sera. It was also very efficient in inhibiting the infectivity of both cercariae and schistosomula. The antigen reactive with the 152-66-9B monoclonal antibody contains two major polypeptides (45 and 30 KD). These polypeptide chains might have originated from the same protein, because they have the same isoelectric point in two-dimensional gel electrophoresis. Moreover, the affinity-purified antigen migrated as only one protein band of approximately 200 KD in SDS-PAGE in nonreducing conditions. The 9B antigen was isolated, purified, and used for immunization, resulting in an antigen dose-dependent partial protection against S. mansoni infection.
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Monoclonal antibodies associated with sodium channel block nerve impulse and stain nodes of Ranvier.
Monoclonal antibodies were generated against native eel electroplax sodium channels in their natural membrane. These antibodies block nerve conduction in rat central (optic) and peripheral (sciatic) nerve. The antibody binding to eel electroplax membrane fragments and to rat brain synaptosomes can be modulated by neurotoxins. Thus it implies that active sites of the sodium channels are immunogenic in their natural membrane. Unlike the antibodies described in the past, our antibodies recognize antigenic determinants which are associated with the physiological activity of the channel and have been conserved through evolution.
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Identification of a surface antigen, common to murine lung and Lewis lung carcinoma, by monoclonal antibody.
Lewis lung carcinoma (3LL) is a spontaneous murine lung carcinoma which preferentially metastasizes into the lung. Monoclonal antibodies (MAb) against 3LL cells were prepared by fusing spleen cells from rats immunized with 3LL and the P3-NSI/I-Ag4 (NSI) plasmacytoma. Two hybridomas were selected which secreted antibodies capable of preferential binding to 3LL cells and not to other malignant cells. Immunohistochemical staining of sections prepared from various mouse organs with one of the MAbs (22.2), revealed exclusive staining of the lung tissue. The data suggest that a lung-specific, 3 LL-associated antigen, is detected by the 22.2 MAb on 3LL cells. The 22.2 antigen is a surface molecule, and is stable following trypsin treatment and fixation by glutaraldehyde or picric acid. Cells that were prepared from a subcutaneous tumor or from lung metastases express high amounts of 22.2 antigen compared to cells of the culture 3LL line (3LLC). Upon reinoculation of 3LLC cells into mice there was a gradual increase in 22.2 antigen expression. It was possible to select from the heterogeneous 3LLC cell population several clones which express high amounts of antigen. These clones differed also in their metastatic patterns. However, the metastatic capacity of these clones did not correlate with expression of the 22.2 antigen. Thus, it appears that expression of the 22.2 antigen is not a necessary condition for formation of lung metastases by 3LL.
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Inhibition of IgE binding to mast cells and basophils by monoclonal antibodies to murine IgE.
In an attempt to identify the site on IgE which binds with high affinity to the Fc epsilon receptor (Fc epsilon R) on mast cells, we established monoclonal anti-IgE antibodies (mAb) by fusion of myeloma cells with rat splenocytes immunized with purified murine IgE mAb. Six individual mAb were found to react with various IgE mAb of different specificities and not with immunoglobulins of other classes. Three different clusters of epitopes on the Fc epsilon portion could be detected by antibody competition studies. These antigenic determinants were expressed on the Fc epsilon portion and required the two heavy chains in their native conformation. Two groups of mAb and their Fab' fragments completely inhibited the binding of 125I-labeled IgE to rat basophilic leukemia cells (RBL), and one mAb inhibited the specific IgE binding only partially (55-65%). Likewise, the Fab' fragments of the purified mAb inhibited the antigen-mediated, IgE-dependent, serotonin release of RBL cells. These in vitro findings were confirmed by in vivo experiments, which demonstrated that the anti-IgE mAb could specifically block passive cutaneous anaphylaxis reaction when injected i.d., before challenging with the antigen. The differences in blocking reactivity of the various anti-IgE mAb are discussed in view of heterogeneity in the IgE-Fc epsilon R interaction.
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Serological analysis of idiotypic determinants on monoclonal antibodies specific to poly(Tyr,Glu)-poly(DLAla)--poly(Lys) and its ordered analogue (Tyr-Tyr-Glu-Glu)-poly(DLAla)--poly(Lys).
Anti-idiotypic sera (aIds) were raised in C57BL/6 mice against monoclonal antibodies (McAbs) which bind poly(Tyr,Glu)-poly(DLAla)--poly(Lys)--abbreviated to (T,G)-A--L--and (Tyr-Tyr-Glu-Glu)-poly(DLAla)--poly(Lys)--abbreviated to (T-T-G-G)-A--L--(nos. 103 and 160) and McAbs which react only with (T-T-G-G)-A--L (nos. 100 and 114). Anti-Id antibodies against 103 McAb reacted with (T,G)-A--L specific antibodies and specifically inhibited their binding to iodinated antigen. Similarly, conventional antibodies against the major idiotypes of (T,G)-A--L-specific antibodies inhibited the binding of 103 McAb to antigen. It is therefore suggested that 103 McAb shares major idiotypes with (T,G)-A--L-specific polyclonal antibodies of C3H.SW origin. Anti-Ids against 114 McAb also inhibited the binding of (T,G)-A--L-specific antibodies to antigen, but the binding of 114 McAb could not be inhibited by conventional aIds. Therefore, idiotypes of 103 and 114 McAbs define idiotypic determinants expressed on two different subpopulations of (T,G)-A--L-specific antibodies: those that carry major idiotypes and those which express idiotypic determinants other than the major one (minor Ids). Anti-idiotypic sera against McAbs nos. 100 and 160 reacted with the homologous idiotypes and not with the major idiotypes of (T,G)-A--L-specific antibodies. In addition to the aforementioned specificities we could define cross-reactive idiotypes (private) shared by McAbs nos. 100, 103 and 114 McAbs nos. 160 and 114. The analysis of idiotypes expressed on anti-(T,G)-A--L McAbs enabled the detection of new antigen binding site related idiotypic determinants in addition to the major idiotypes which were immunodominant in the polyclonal anti-(T,G)-A--L antibodies.
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Monoclonal anti-VH antibodies recognize a common VH determinant expressed on immunoglobulin heavy chains from various species.
Our previous work using rabbit antibodies to the variable region of MOPC315 myeloma heavy chain (VH) has indicated the existence of framework determinant(s) common to many murine heavy chains. Here we report the characterization of anti-VH monoclonal antibodies (mAb) prepared in an attempt to elucidate the nature of the common VH determinant. We immunized AKR/J mice with a purified VH315 fragment and generated somatic cell hybrids by the fusion of the immune AKR/J splenocytes with the NS1 myeloma cells. Thirty-seven common anti-VH and 57 subgroup VHI-specific hybridomas have been established and characterized. Whereas the anti-subgroup mAb seemed to react with a determinant unique to the MOPC315 (mouse VHI) subgroup, all the anti-VH mAb reacted with myeloma heavy chains of different VH subgroups, class and allotypes. Antibody competition studies revealed that the VH subgroup determinants are distinct from the common VH determinants and that both were also recognized by the rabbit polyclonal antibodies. The common VH determinants were found to be "hidden" determinants on intact immunoglobulin molecules being exposed only on isolated heavy chains. Furthermore, they are sequential determinants since they are preserved on fully denatured heavy chains. The common VH determinants are shared by immunoglobulins of a wide range of vertebrates from amphibia to man and thus represent antigenic structures which were highly conserved throughout evolution.
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Anti-schistosome monoclonal antibodies of different isotypes--correlation with cytotoxicity.
Five monoclonal antibodies specific towards Schistosoma mansoni antigens were prepared by fusion of spleen cells of infected and immunized mouse with the murine myeloma NS-1 cells. Three of the five antibodies belonged to the IgG1 class, one was an IgM and the fifth one was an IgE. The IgE monoclonal antibody designated 54.10, induced antigen-specific degranulation of rat basophilic cell line, a property which served as the basis for the screening assay. Its biological function was demonstrated by a specific macrophage activation that led to killing of schistosomula; no such killing was obtained with anti-schistosome antibodies of other classes or with IgE of different antigenic specificity. The second monoclonal antibody of biological significance was an IgG1, designated 27.21 which is reactive in the immunofluorescence staining of surface antigens on intact schistosomula. All three monoclonal antibodies that belonged to the IgG1 class were effective in mediating killing of schistosomula by complement, with the highest effect exerted by 27.21. It is thus apparent that the 27.21 monoclonal antibody is directed against a densely distributed surface antigen on the schistosomula membrane which is possibly involved in the protective immunity. Preliminary data showed that immunoprecipitation with the 27.21 antibodies results in the isolation of three major protein bands, of 60 kd, 50 kd, 19 kd, respectively.
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