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Biomedical subjects

Z Eshhar

Publications and source records attributed to Z Eshhar.

At least 19 recordsLinked to original sources
PubMed1992

Detection of catalytic monoclonal antibodies.

Several laboratories have now shown that monoclonal antibodies having enzyme-like properties can be generated. The generation of catalytic antibodies makes use of the same basic procedures that have been used for the generation of binding monoclonal antibodies, yet the process involves an additional crucial step: screening for catalytic activity. In this paper we address the unique problems involved in the detection of inefficient catalytic activity that is accompanied by uncatalyzed background reaction. An analysis that allows optimization of assay conditions and estimation of the minimal antibody concentration required to observe catalysis is presented. The results indicate that the structure of the substrate should be optimized to increase its affinity (i.e., decrease its Km) and reduce its concentration to pseudo-first-order conditions (S(O) much less than Km) so that the signal observed in the presence of a catalytic antibody (delta Pcat) is significantly higher than that of the background (delta P(uncat)). Other factors involved in the screening procedures, e.g., sensitivity of the assay, solubility and reactivity of the substrate, and purity of the antibody preparation, are also discussed. The effect of these assay parameters on the ability to detect catalytic activity is demonstrated with p-nitrophenyl ester-hydrolyzing antibodies.

Animals↗
PubMed1992

The human mast cell receptor binding site maps to the third constant domain of immunoglobulin E.

The characterization of the site on the IgE molecule which accommodates the high affinity receptor for IgE (Fc epsilon RI) should allow the design of IgE analogues which can be utilized to block allergic responses. Using chimeric human IgE molecules in which different constant region domains were exchanged with their murine homologues, we demonstrate here that the C epsilon 3 in its native configuration is essential for the binding to the alpha subunit of the human Fc epsilon RI. Deletion of the human C epsilon 2 from such chimeric molecules did not impair their ability to interact with the Fc epsilon RI, indicating that C epsilon 2 is not directly involved in the human Fc epsilon RI binding site and that C epsilon 3 alone is necessary and sufficient to account for most of the human Fc epsilon RI-binding capacity.

Animals↗
PubMed1992

Endowing T cells with antibody specificity using chimeric T cell receptors.

T cells recognize antigen in the form of a peptide associated with a cell surface molecule encoded by the major histocompatibility gene complex (MHC). The elaborate requirements for the T cell receptor (TCR)-antigen interaction stand in contrast to the simple and defined nature of the antigenic determinants recognized by antibodies. The similarity in the molecular structure and gene organization between antibodies and the TCR has prompted attempts to interchange the antigen-binding, variable regions of these molecules. To this end, chimeric TCR (cTCR) genes, composed of the variable domains of antibodies linked to TCR constant regions, have been used to confer antibody-type specificity on T cells. cTCR-expressing T cells respond to stimulator cells as well as to immobilized antigen in an MHC unrestricted and independent manner. The antibody-like specificity of the resulting T cells has been exploited, using defined ligands, to elucidate the physicochemical parameters that govern TCR-mediated signaling, and to provide a useful experimental system to study the role of MHC and cell-adhesion/accessory molecules in T cell activation. The successful expression of such cTCR in transgenic mice opens new avenues to explore the role of the MHC in T cell development and maturation. Eventually, chimeric receptors specific to tumor or viral antigens might be used for in vivo targeting of T cells in the framework of immuno- and gene therapy.

Animals↗
PubMed1992

Functional assembly of chimeric T-cell receptor chains.

We have generated cytotoxic T-cell hybridomas expressing chimeric T-cell receptors (cTCR) with an antibody-type specificity for the TNP hapten. Transfectants expressing the cTCR genes could mediate specific lysis of haptenated tumor cell lines of various types and secrete IL-2 upon stimulation with TNP modified cells. In a previous report, we showed that double-gene transfectants expressing either VHC alpha and VLC beta or VHC beta and VLC alpha could be activated by TNP-modified stimulator cells or TNP proteins immobilized on plastic. Single-chain transfectants (expressing VHC alpha or VHC beta alone) could be mainly activated by TNP-cells. We now report that transfection of chimeric VHC alpha gene into an alpha-chain-defective mutant restores the surface expression of the TCR/CD3 complex. In parallel, such transfectants regained the ability to respond to mitogen and anti-CD3 antibodies and responded weakly to TNP cells. Double gene transfectants, bearing 2 complementary chimeric chains, expressed high amounts of cTCR on their surface, sufficient to acquire sound anti-TNP reactivity. Cells expressing the VHC beta gene only were not functional and had no detectable surface TCR chains. Taken together, our results suggest that chimeric VHC alpha chains can pair with endogenous V beta C beta chains, but that there is preferential association between complementary chimeric chains, resulting in higher functional expression of the chimeric TCR.

Animals↗
PubMed1991

Engraftment and development of human T and B cells in mice after bone marrow transplantation.

A model for human lymphocyte ontogeny has been developed in a normal mouse. Human bone marrow, depleted of mature T and B lymphocytes, and bone marrow from mice with severe combined immunodeficiency were transplanted into lethally irradiated BALB/c mice. Human B and T cells were first detected 2 to 4 months after transplantation and persisted for at least 6 months. Most human thymocytes (30 to 50 percent of total thymocytes) were CD3+CD4+CD8+. Human immunoglobulin was detected in some chimeras, and a human antibody response to dinitrophenol could be generated after primary and secondary immunization.

Animals↗
PubMed1991

CD4 and CD8 accessory molecules function through interactions with major histocompatibility complex molecules which are not directly associated with the T cell receptor-antigen complex.

Both the subset-specific, CD4 and CD8 T cell accessory molecules and the antigen-specific T cell receptor (TcR) interact with major histocompatibility complex (MHC) class I and class II molecules on the surface of antigen-presenting cells. We analyzed whether the CD4/CD8 molecules exert their accessory function through binding with the same MHC molecules which participate in the TcR-antigen-MHC complex. We utilized a CD4-, CD8-, class I-allospecific T cell hybridoma which functionally manifests both cytotoxic T lymphocyte (CTL) and T helper1 (Th1) phenotypes, and rendered it bispecific by transfecting it with genes encoding either a class II-restricted, 2,4,6-trinitrophenyl (TNP)-I-Ad-specific TcR or a non-MHC-restricted chimeric TcR, composed of a variable part of an anti-TNP antibody. Expression of either CD4 or CD8 transgenes in these hybridomas enhanced and augmented their reactivity towards the appropriate target cells regardless of the type of TcR-MHC interaction. Thus, class I-specific responses could be enhanced through CD4-class II interactions, and class II-restricted responses could be augmented through CD8-class I interactions. Furthermore, these accessory molecules also potentiated TNP-specific responses by the chimeric TcR which is MHC unrestricted. The accessory molecules facilitated both interleukin 2 (IL2) production and cytolytic activity by shortening the activation time and rendering the cells responsive to lower antigenic stimuli. The degree of activity of the T cell hybridomas correlated with the level of accessory molecule expression and was not related to the effector function mediated by the cells. Anti-CD4 or -CD8 antibodies completely inhibited the activity of transfectants expressing the corresponding accessory molecule, regardless of the MHC type of the TcR interaction. Such antibodies blocked direct TcR stimulation provided by either anti-T3/Ti antibodies or lectins, but could not inhibit the activation through agents that bypass the TcR such as phorbol 12-myristate 13-acetate plus ionophore. Taken together, these studies demonstrate that the CD8/CD4 molecules can exert their accessory function through interactions with MHC molecules which are not directly associated with the TcR-Ag-MHC complex, and that this accessory effect is associated with TcR-mediated triggering at an early stage of the signaling process and is not related to the effector mechanism assigned to the CD4 and CD8 T cell subsets.

Animals↗
PubMed1991

Mapping of the high affinity Fc epsilon receptor binding site to the third constant region domain of IgE.

Identification of the precise region(s) on the IgE molecule that take part in the binding of IgE to its high affinity receptor (Fc epsilon RI) may lead to the design of IgE analogues able to block the allergic response. To localize the Fc epsilon RI-binding domain of mouse IgE, we attempted to confer on human IgE, which normally does not bind to the rodent receptor, the ability to bind to the rat Fc epsilon RI. Employing exon shuffling, we have expressed chimeric epsilon-heavy chain genes composed of a mouse (4-hydroxy-3-nitrophenyl)acetic acid (NP)-binding VH domain, and human C epsilon in which various domains were replaced by their murine counterparts. This has enabled us to test the Fc epsilon RI-binding of each mouse IgE domain while maintaining the overall conformation of the molecule. All of the chimeric IgE molecules which contain the murine C epsilon 3, bound equally to both the rodent and human receptor, as well as to monoclonal antibodies recognizing a site on IgE which is identical or very close to the Fc epsilon RI binding site. Deletion of the second constant region domain did not impair either the binding capacity of the mutated IgE or its ability to mediate mast cell degradation. These results assign the third epsilon domain of IgE as the principal region involved in the interaction with the Fc epsilon RI.

Animals↗
PubMed1990

Simple method for selecting catalytic monoclonal antibodies that exhibit turnover and specificity.

Monoclonal antibodies were raised against a mono-p-nitrophenyl phosphonate ester to elicit catalytic antibodies capable of hydrolyzing the analogous p-nitrophenyl ester or carbonate. Potential catalytic antibody producing clones were selected, by use of a competitive inhibition assay, on the basis of their affinity for a "short" transition-state analogue, a truncated hapten which maximizes the relative contribution of the transition-state structural elements to binding. Of 30-40 clones that would have been examined on the basis of hapten binding alone, 7 were selected and 4 of these catalyzed the hydrolysis of the relevant p-nitrophenyl ester. This competitive inhibition technique represents a general approach for selecting potential catalytic antibodies and significantly increases the probability of obtaining efficient catalytic monoclonal antibodies. Further study of the catalytic antibodies revealed significant rate enhancement (kcat/kuncat approximately 10(4)) and substrate specificity for the hydrolysis of the analogous ester and, for three of the antibodies, of the analogous carbonate. The antibodies displayed turnover, an essential feature of enzymes. Evidence that catalysis occurred at the antibody combining sites was provided by the identity of the binding and the catalysis-inhibition specificity patterns.

Antibodies, Monoclonal↗
PubMed1990

Human monoclonal antibody derived from an autoimmune thrombocytopenic purpura patient, recognizing an intermediate filament's determinant common to vimentin and desmin.

Human monoclonal antibody (mAb) technology has been helpful in identifying autoantibodies that are involved in various autoimmune disorders. We report here the results of such an attempt to immortalize antibody-forming cells from spleen of an autoimmune thrombocytopenic purpura (ATP) patient and characterize the resulting mAb. The human mAb we derived, denoted (4G9), binds to the cytoskeletal network. Using immunofluorescence analyses of permeabilized and fixed cell lines and tissues, the 4G9 mAb was shown to be anti-vimentin specific by virtue of its intracellular staining pattern, decoration of cell lines of mesenchymal (but not epithelial) origin, and by the fact that polyclonal anti-vimentin (and not anti-actin, prekeratin, tubulin or vinculin) antibodies inhibited its binding to intermediate filaments of a fibroblastoid cell line. The presence of vimentin in platelets was also confirmed in the present study by immunoblotting of platelet extract using murine anti-vimentin mAb. Interestingly, in addition to vimentin the 4G9 mAb decorated intermediate filaments in desmin-expressing muscular cells, suggesting that the 4G9 epitope is most likely located within the homologous sequences that are known to be shared between vimentin and desmin.

Animals↗
PubMed1990

Monoclonal antibodies against carcinoembryonic antigen (CEA) discriminate between CEA produced by gastrointestinal tumors and CEA produced by other tumors.

Carcinoembryonic antigen (CEA) is a common tumor marker. It is not specific since many tumor types express it. We report here the use of a series of monoclonal anti-CEA antibodies with apparent restricted activity. These antibodies reacted histochemically with CEA produced by tumors originating in the gastrointestinal tract, but they did not react with CEA-secreting tumors of nongastrointestinal origin. We have used this phenomenon to accurately diagnose the tumor of origin in 20 patients who had a CEA-producing metastatic tumor, but in whom the primary origin was clinically not yet discovered. The use of such antibodies for diagnostic purposes may be an adjunct to other methods in evaluating tumors of unknown origin.

Aged↗
PubMed1989

Mapping of murine IgE epitopes involved in IgE-Fc epsilon receptor interactions.

The generation of anti-IgE monoclonal antibodies has permitted the identification of various serological epitopes on the IgE molecule. The relationship of the sites on IgE recognized by such antibodies to the Fc epsilon receptor (Fc epsilon R) interaction site has been determined using cross-inhibition studies. However, interpretation of this type of experiment is limited by problems of steric hindrance. Thus, to accomplish precise mapping on the IgE molecule of the Fc epsilon R interaction site and the binding sites of various anti-IgE mAb, we employed site-directed mutagenesis of the IgE heavy chain gene. To this end we have constructed and expressed a recombinant murine constant epsilon heavy chain (C epsilon) gene bearing a (4-hydroxy-3-nitrophenyl)acetic acid (NP)-binding VH region. Several site-specific mutants in the C epsilon 3 and C epsilon 4 domains of this recombinant C epsilon gene were prepared and expressed by transfection into the light chain-producing J558L myeloma cell line. The resulting IgE antibodies were tested for binding to mast cells and to various anti-IgE mAb. The mutants produced include a proline to histidine point mutant at amino acid residue 404 in the C epsilon 3 domain, a mutant with a truncated C epsilon 4 domain, a mutant with a 45 amino acid deletion in the carboxy end of C epsilon 3, and a chimeric human C epsilon in which the human C epsilon 3 was replaced by the homologous mouse C epsilon 3 domain. These mutants have permitted the localization, to the C epsilon 3 domain, of the epitopes recognized by the 84.1C and 95.3 anti-IgE mAb. The 84.1C mAb recognizes a site on IgE which is identical or very close to the Fc epsilon R binding site, and 95.3 recognizes a site on IgE which is related, but not identical to the Fc epsilon R binding site. The antigenic determinant recognized by the 51.3 mAb, which is inefficient at blocking the IgE-Fc epsilon R interaction, has been mapped to the C epsilon 4 domain. When tested for binding to the Fc epsilon R on RBL-2H3 cells, the point mutant bound to the Fc epsilon R with twofold reduced affinity, while the C epsilon 3 deletion mutant and the mutant truncated in C epsilon 4 lost all receptor binding activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1989

Expression characteristics of two potential T cell mediator genes.

T lymphocyte subset-specific cDNA clones were recently isolated by a modified differential screening procedure. The expression patterns of two of these cDNAs, designated as 4-1BB and L2G25B, were studied in greater detail. Nucleotide sequence comparison revealed that 4-1BB was not previously recognized. Although the L2G25B sequence had been recognized recently, the function of the encoded molecule has yet to be well studied. The transcripts of the two cDNAs were inducible by concanavalin A in mouse spleen cells, cloned helper T cells (L2), cloned cytolytic T cells (L3), and cytolytic T cell hybridomas. They were also inducible with stimulation through antigen receptor (TCR), with immobilized anti-TCR antibody in cloned T cells L2, dB45, and L3. Concanavalin A inducibility was inhibited by cyclosporin A. They were not inducible by IL-2 stimulation. The expression patterns of these transcripts were similar to those of IFN-gamma, except that the level of transcripts of the two cDNAs was at least fivefold lower than that of IFN-gamma, and the peak level of expression occurred earlier. These data suggest that L2G25B and 4-1BB may represent new T cell mediators.

Animals↗
PubMed1989

Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity.

To design and direct at will the specificity of T cells in a non-major histocompatibility complex (MHC)-restricted manner, we have generated and expressed chimeric T-cell receptor (TcR) genes composed of the TcR constant (C) domains fused to the antibody's variable (V) domains. Genomic expression vectors have been constructed containing the rearranged gene segments coding for the V region domains of the heavy (VH) and light (VL) chains of an anti-2,4,6-trinitrophenyl (TNP) antibody (SP6) spliced to either one of the C-region gene segments of the alpha or beta TcR chains. Following transfection into a cytotoxic T-cell hybridoma, expression of a functional TcR was detected. The chimeric TcR exhibited the idiotope of the Sp6 anti-TNP antibody and endowed the T cells with a non-MHC-restricted response to the hapten TNP. The transfectants specifically killed and produced interleukin 2 in response to TNP-bearing target cells across strain and species barriers. Moreover, such transfectants responded to immobilized TNP-protein conjugates, bypassing the need for cellular processing and presentation. In the particular system employed, both the TNP-binding site and the Sp6 idiotope reside almost exclusively in the VH chain region. Hence, introduction into T cells of TcR genes containing only the VHSp6 fused to either the C alpha or C beta was sufficient for the expression of a functional surface receptor. Apparently, the VHC alpha or VHC beta chimeric chains can pair with the endogenous beta or alpha chains of the recipient T cell to form a functional alpha beta heterodimeric receptor. Thus, this chimeric receptor provides the T cell with an antibody-like specificity and is able to effectively transmit the signal for T-cell activation and execution of its effector function.

Animals↗
PubMed1989

Monoclonal antibodies for affinity purification of IL-6/IFN- beta 2 and for neutralization of HGF activity.

Two types of recombinant human IL-6 (rIL-6) were used for the development of specific monoclonal antibodies. The first was produced in E. coli and used for immunization, the second was produced in Chinese Hamster Ovary Cells (CHO) and used for screening. The complete translated sequence of the cDNA coding for human IL-6 was fused, in phase, to protein-A and the hybrid gene was fused to the strong lambda PR promoter. This protein was purified from bacterial extracts by chromatography on rabbit IgG-Sepharose columns. After six injections of the purified protein into mice, sera were tested for their binding titer in a solid phase radioimmunassay (sRIA) and for the specificity of binding by Western blots. In the sRIA, crude supernatants of CHO cells (harboring a plasmid containing the human IL-6 gene and expressing high levels of IL-6 but no protein-A or any bacterial antigen) were bound to a solid support, reacted with supernatants of the hybridomas and finally detected with [125I]-goat anti-mouse antibodies. Spleen cells derived from a mouse showing the highest binding titer were fused to mouse myeloma cells. The hybridomas were screened by the sRIA and several positive clones were isolated and characterized. One of the clones was found to neutralize the hybridoma growth factor activity of the rIL-6 from both sources. The same clone was also used for Western blots and for affinity purification of both natural and recombinant IL-6 (E. coli and CHO).

Animals↗
PubMed1989

Monoclonal antibodies to the human interferon-gamma receptor: blocking of the biological activities of interferon-gamma and purification of the receptor.

Monoclonal antibodies against the human interferon-gamma (IFN-gamma) receptor were developed by injecting mice with a preparation of receptor that was purified from solubilized placental membranes by ligand affinity chromatography. Three antibodies were identified by their ability to block the binding of 125I-labeled IFN-gamma to its receptor on HeLa cells at 4 degrees C. One of these antibodies blocked several biological activities of IFN-gamma, including its antiviral activity, its ability to induce HLA-DR surface antigens, and its ability to protect cells from NK cell-mediated cytotoxicity. This antibody exhibited higher binding capacity to cells at 37 degrees C and was significantly less displaceable by an excess of IFN-gamma as compared with the other two antibodies. Immunoaffinity chromatography of solubilized crude placental membrane preparation yielded a purified receptor that exhibited a molecular weight of 88,000. The purified receptor retained its ability to bind 125I-labeled IFN-gamma in solution.

Animals↗