The T-body approach: potential for cancer immunotherapy.
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Animals↗
Biomedical subjects
Z Eshhar
Publications and source records attributed to Z Eshhar.
Prolonged inhibition of IgE production in mice following treatment with an IgE-specific immunotoxin.
The synthesis of IgE antibodies by B cells is the first in a series of steps resulting in an allergic response. To eliminate IgE-bearing B cells and thereby prevent IgE production, we have developed an immunotoxin (ITA) composed of the non-anaphylactic 84.1c anti-mouse IgE mAb and the A chain of ricin (ricin A). This ITA specifically inhibited the induction of IgE synthesis by lipopolysaccharide plus interleukin-4 (LPS + IL-4) in vitro, and antigen-specific IgE production in vivo in adult mice. A single dose of anti-IgE ITA, given within a week (either before or after) of antigen challenge completely abolished antigen-specific primary IgE responses. No IgE production was seen for 2 months after ITA treatment. Following antigenic re-challenge, a suppressed secondary response (over 50% reduction) was still seen in the ITA-treated mice, 100 days after immunization. The results of this study demonstrate the potential use of anti-IgE toxin conjugates for the suppression of periodic (seasonal) allergic outbreaks.
Animals↗
Crystal structure of the complex of a catalytic antibody Fab fragment with a transition state analog: structural similarities in esterase-like catalytic antibodies.
The x-ray structure of the complex of a catalytic antibody Fab fragment with a phosphonate transition-state analog has been determined. The antibody (CNJ206) catalyzes the hydrolysis of p-nitrophenyl esters with significant rate enhancement and substrate specificity. Comparison of this structure with that of the uncomplexed Fab fragment suggests hapten-induced conformational changes: the shape of the combining site changes from a shallow groove in the uncomplexed Fab to a deep pocket where the hapten is buried. Three hydrogen-bond donors appear to stabilize the charged phosphonate group of the hapten: two NH groups of the heavy (H) chain complementarity-determining region 3 (H3 CDR) polypeptide chain and the side-chain of histidine-H35 in the H chain (His-H35) in the H1 CDR. The combining site shows striking structural similarities to that of antibody 17E8, which also has esterase activity. Both catalytic antibody ("abzyme") structures suggest that oxyanion stabilization plays a significant role in their rate acceleration. Additional catalytic groups that improve efficiency are not necessarily induced by the eliciting hapten; these groups may occur because of the variability in the combining sites of different monoclonal antibodies that bind to the same hapten.
Antibodies, Catalytic↗
In vivo antitumor activity of T cells redirected with chimeric antibody/T-cell receptor genes.
In an effort to broaden the applicability of adoptive cellular immunotherapy toward nonmelanoma cancers, we have designed chimeric antibody/T-cell receptor genes composed of the variable domains from mAbs joined to T-cell receptor-signaling chains. We have demonstrated that T cells retrovirally transduced with these genes can recognize antibody-defined antigens and that this recognition leads to T-cell activation, specific lysis, and cytokine release. In this study, we have examined the in vivo activity of murine T cells transduced with a chimeric receptor gene (MOv-gamma) derived from the mAb MOv18, which binds to a folate-binding protein overexpressed on most human ovarian adenocarcinomas. Nude mice that were given i.p. implants of human ovarian cancer (IGROV) cells were treated 3 days later with i.p. murine tumor-infiltrating lymphocytes (TIL) derived from an unrelated tumor. Mice treated with MOv-gamma-transduced TIL (MOv-TIL) had significantly increased survival compared to mice treated with saline only, nontransduced TIL, or TIL transduced with a control anti-trinitrophenyl chimeric receptor gene (TNP-TIL). In another model, C57BL/6 mice were given i.v. injections of a syngeneic methylcholanthrene-induced sarcoma transduced with the folate-binding protein (FBP) gene. Three days later, mice were treated i.v. with various transduced murine TIL (derived from an unrelated tumor), followed by low-dose systemic interleukin 2. Eleven days after tumor injection, mice were sacrificed, and lung metastases were counted. In multiple experiments, mice receiving MOv-TIL had significantly fewer lung metastases than did mice treated with interleukin 2 alone, nontransduced TIL, or TNP-TIL. These studies indicate that T cells can be gene modified to react in vivo against tumor antigens, defined by mAbs. This approach is potentially applicable to a number of neoplastic and infectious diseases and may allow adoptive immunotherapy against types of cancer not previously amenable to cellular immunotherapy.
Animals↗
Unexpectedly high occurrence of catalytic antibodies in MRL/lpr and SJL mice immunized with a transition-state analog: is there a linkage to autoimmunity?
Upon testing the ability of several strains of mice to elicit esterolytic antibodies after immunization with a p-nitrobenzyl phosphonate hapten, we have found that the occurrence of catalytic antibodies in SJL and MRL/lpr autoimmune mice is dramatically higher than in normal mouse strains (e.g., the wild-type MRL/++ or BALB/c). Fewer than 10 catalytic clones are usually obtained from a single fusion of lymphocytes taken from normal mice, whereas several hundred catalytic clones are obtained in SJL or MRL/lpr mice. Differences in the numbers of hapten-binding clones do not account for the high occurrences of catalytic clones in these strains. This phenomenon prevailed in the early responses; in both SJL and MRL/lpr mice a significant decline in the appearance of catalytic clones was observed after multiple immunizations. Esterolytic antibodies were not found in MRL/lpr mice immunized with haptens that do not mimic the transition state for the hydrolysis of the ester substrate (e.g., with a substrate analog). The catalytic antibodies manifest high specificity to the antigen and variability in their binding and catalytic properties. The use of autoimmunity-prone mice may greatly expand the repertoire of catalytic clones elicited against a transition-state analog hapten. More intriguing is the possible linkage between autoimmunity and the appearance of catalytic antibodies. These results suggest that there is normally a selection against the expression of certain variable genes encoding antibodies with catalytic activity.
Animals↗
Specific elimination of IgE production using T cell lines expressing chimeric T cell receptor genes.
B cells that are destined to secrete IgE express a membrane-bound form of IgE (mIgE) on their cell surface. Thus, elimination of such mIgE-positive cells should result in the suppression of IgE production, thereby alleviating the symptoms of IgE-mediated allergy. In this study, we examined, in a model system, whether IgE-specific effector T cells can be used specifically to eradicate IgE-producing B cells. To this end, we endowed T cells with anti-IgE specificity using chimeric T cell receptors (cTCR) containing the variable region domain (Fv) of the 84.1c non-anaphylactic anti-mouse IgE monoclonal antibody (mAb). Two configurations of chimeric receptor were used: in the first, we combined the heavy and light variable region chains of 84.1c with the constant (C) regions of the TCR alpha and beta chains. The second construct consisted of a chimeric single-chain receptor (scFvR), composed of a single-chain Fv region of the 84.1c antibody and the C beta domain of the TCR. Following transfection of the cTCR or the scFvR genes into the murine MD.45 cytotoxic T cell hybridoma or the Jurkat human T cell line, functional expression of IgE-specific chimeric receptors was detected on the cell surface. The transfected cells secreted interleukin-2 upon stimulation with immobilized IgE or fixed IgE-producing hybridoma cells. Moreover, cytotoxic T cell hybridomas expressing the chimeric receptor genes specifically eliminated IgE-secreting B cells in vitro, resulting in isotype-specific suppression of IgE production.
Animals↗
Chimaeric T-cell receptors specific to a B-lymphoma idiotype: a model for tumour immunotherapy.
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Animals↗
Calpain (Ca(2+)-dependent thiol protease) in erythrocytes of young and old individuals.
Limited proteolysis by calpain (Ca(2+)-activated protease; EC 3.4.22.17) is believed to regulate the function of membrane enzymes and modify the behavior of membrane structural proteins. Calpain is activated by autolysis. The degradation of band 3 protein by mu-calpain is known to be enhanced in erythrocyte membranes from human individuals > 70 years old (old) as compared with that from individuals 20-30 years old (young). In the present study, monoclonal antibody to mu-calpain was used to study the behavior of calpain in erythrocytes of young and old individuals. Less calpain was found in erythrocyte cytosol and membranes from old than in those from young. Increasing the erythrocyte Ca2+ induced translocation of calpain to the cell membrane and autolysis of the enzyme. Alkylation of erythrocyte thiols also promoted translocation of calpain to the membrane, especially in the presence of Ca2+. When calpain was added to erythrocyte membranes, initial binding was greater and subsequent autolysis faster in old than in young individuals, possibly arising from alterations in cell membranes of old individuals. The enhanced calpain autolysis was accompanied by enhanced degradation of band 3 protein in the old. The results suggest that calpain in old individuals is translocated to the cell membrane and is activated by autolysis, resulting in degradation of certain membrane proteins and loss of calpain. Enhanced calpain-induced membrane proteolysis may play a role in abnormal cell destruction (e.g., shortening the life span of erythrocytes in the aged, neuronal degeneration, etc). The erythrocyte membrane provides a convenient model for the study of age-associated alterations in cell membranes and in calpain behavior.
Adult↗
Crystal structure of a catalytic antibody Fab with esterase-like activity.
BACKGROUND: Antibodies with catalytic properties can be prepared by eliciting an antibody response against 'transition state analog' haptens. The specificity, rate and number of reaction cycles observed with these antibodies more closely resemble the properties of enzymes than any of the many other known enzyme-mimicking systems. RESULTS: We have determined to 3 A resolution the first X-ray structure of a catalytic antibody Fab. This antibody catalyzes the hydrolysis of a p-nitrophenyl ester. In conjunction with binding studies in solution, this structure of the uncomplexed site suggests a model for transition state fixation where two tyrosines mimic the oxyanion binding hole of serine proteases. A comparison with the structures of known Fabs specific for low molecular weight haptens reveals that this catalytic antibody has an unusually long groove at its combining site. CONCLUSION: Since transition state analogs contain elements of the desired product, product inhibition is a severe problem in antibody catalysis. The observation of a long groove at the combining site may relate to the ability of this catalytic antibody to achieve multiple cycles of reaction.
Antibodies, Monoclonal↗
Functional expression in mast cells of chimeric receptors with antibody specificity.
Chimeric genes composed of a single-chain Fv domain (scFv) of an antibody linked with receptor chains normally present in cells of hematopoietic origin were constructed. Such genes could be expressed as functional surface receptors in the RBL-2H3 (rat basophilic leukemia) mast cell line. The chimeric receptors exhibited binding properties of an antibody molecule and triggered degranulation of transfected mast cells on stimulation with antigen. Genetically engineered designer cells (e.g., T-lymphocytes, mast cells, or natural killer cells), equipped with built-in antibody-type recognition, can be now exploited for immunotherapy.
Amino Acid Sequence↗
Catalytic antibodies: a critical assessment.
In the past few years, antibodies that catalyze a variety of reactions with enzyme-like properties have been produced. The present review is of a critical nature, rather than a survey or an introduction to the field of catalytic antibodies. Here, we examine the performance of catalytic antibodies in light of the features that define an enzyme: substrate specificity, rate enhancement, and turnover. We also refer to some limitations of the technologies currently used for their generation. In the future, antibodies may provide a new repertoire of tailor-made, enzyme-like, catalysts with possible applications in biology, medicine, and biotechnology. In the following sections, we emphasize that these applications will require far more efficient catalysts than are presently available, and we point to several trends for future research that may offer more efficient catalytic antibodies.
Animals↗
Differences in the biochemical properties of esterolytic antibodies correlate with structural diversity.
A prerequisite to the design and engineering of catalytic antibodies is the knowledge of their structure and in particular which residues are involved in binding and catalysis. We compared the structure and catalytic properties of a series of six monoclonal antibodies which were all raised against a p-nitrophenyl (PNP) phosphonate and which catalyze the hydrolysis of p-nitrophenyl esters. Three of the antibodies (Group I) have similar light and heavy chain variable regions. The other three antibodies have similar VL regions of which two (Group II) have VH regions from the MOPC21 gene family and the remaining one (Group III) a VH from the MC101 gene family making a total of three different groups based on their V region sequences. The structural division into groups is paralleled by the differences in binding constants to hapten analogs, substrate specificity and the susceptibility of the catalytic activity of the antibodies to chemical modification of tryptophan and arginine residues. The relative binding of a transition state analog to the binding of substrate is much higher for the Group I antibodies than for the other groups. Only the Group I antibodies can catalyze the hydrolysis of a carbonate substrate. However all of the antibodies lose catalytic activity upon specific tyrosine modification which highlights the importance of tyrosine in the active site of the antibodies. Thus, antibodies raised against a single hapten can give antibodies with different structures, and correspondingly different specificities and catalytic properties.
Amino Acid Sequence↗
pH on-off switching of antibody-hapten binding by site-specific chemical modification of tyrosine.
Tetranitromethane (TNM) chemically mutates the binding sites of antibodies so that the nitrated antibodies exhibit pH-dependent binding near physiological pH. Three monoclonal antibodies were selectively modified, each under different conditions, with the resultant loss of binding activity at pH > 8 which is recovered at pH < 6. Recovery and loss of binding are ascribed to the protonation and deprotonation, respectively, of the hydroxyl group of the resulting 3-nitrotyrosine side chain (pKa approximately 7) at the binding site of these antibodies. pH on-off dependency of binding activity, common to all TNM-modified antibodies studied by us so far, may find use in a variety of applications in which controlled modulation under mild conditions is required.
2,4-Dinitrophenol↗
Targeting of T lymphocytes to Neu/HER2-expressing cells using chimeric single chain Fv receptors.
Cell surface molecules essential for the transformed phenotype or growth of malignant cells are attractive targets for anticancer immunotherapy. Antibodies specific to Neu/HER2, a human adenocarcinoma-associated growth factor receptor, were demonstrated to have tumor-inhibitory capacity. Yet, the inefficient accessibility of antibodies to solid tumors limits their clinical use. To redirect effector lymphocytes to adenocarcinomas, we constructed and functionally expressed in T cells chimeric single chain receptor genes incorporating both the Ag-binding domain of anti-Neu/HER2 antibodies and the zeta-signal-transducing subunit of the TCR/CD3 complex or the gamma-signal-transducing subunit of the Ig Fc receptor complex. Surface expression of the anti-Neu/HER2 chimeric genes in cytotoxic T cell hybridomas endowed them with specific Neu/HER2 recognition enabling their activation for IL-2 production and lysis of transformed cells overexpressing Neu/HER2. These chimeric genes hold promise for the immunotherapy of cancer.
Animals↗
Lysis of ovarian cancer cells by human lymphocytes redirected with a chimeric gene composed of an antibody variable region and the Fc receptor gamma chain.
To expand the spectrum of recognition of effector lymphocytes and to redirect them towards predefined targets, we have altered the specificity of human tumor-infiltrating lymphocytes (TIL) through stable modification with chimeric receptor genes consisting of single-chain antibody variable regions linked to the gamma subunit common to the immunoglobulin (Ig)G and IgE Fc receptors. Using either hapten or ovarian carcinoma-specific monoclonal antibodies, we constructed chimeric receptor genes and retrovirally introduced them into CD8+ TIL. Redirected TIL specifically lysed trinitrophenyl-labeled Daudi or a human ovarian carcinoma cell line (IGROV-1), and secreted granulocyte/macrophage colony-stimulating factor upon stimulation with the appropriate antigen. This strategy may allow new approaches towards the adoptive immunotherapy of cancer in humans.
Cloning, Molecular↗
Fine specificity of the IgE interaction with the low and high affinity Fc receptor.
The characterization of the site(s) on the IgE molecule that accommodate the high (Fc epsilon RI) and low (Fc epsilon RII) affinity receptors for IgE should allow the design of IgE analogues that can be used to block the onset of the allergic response or to regulate IgE production. To identify the IgE domain responsible for receptor binding, we generated a series of chimeric IgE antibodies in which constant region domains were interchanged between the human and mouse molecules. Binding studies with these chimeras revealed that both the high and low affinity receptor binding-sites reside primarily in the third constant domain of IgE (C epsilon 3). Additional chimeric IgE molecules were generated in which different parts of the human C epsilon 3 domain were exchanged with their murine homologues. Binding experiments with these chimeras suggest that not only the sequence of a particular C epsilon 3 fragment, but the entire C epsilon 3 domain in its native configuration is essential for binding to the Fc epsilon RI. The amino acid residues determining the species specificity of the Fc epsilon RII are not contained in the first 16 amino acids of the C epsilon 3 domain.
Animals↗
catELISA: a facile general route to catalytic antibodies.
The low abundance and activity of catalytic antibodies are major obstacles to their selection from the virtually unlimited repertoire of antibody binding sites. The requirement for new screening methodologies is further emphasized by the availability of combinatorial libraries, in which a functional polypeptide has to be selected out of millions of possibilities. We present a simple and sensitive screening approach (termed catELISA) based on immobilized substrates and immunodetection of the end product of the catalyzed reaction. The feasibility of catELISA is demonstrated here by the generation of potent ester-hydrolyzing antibodies by direct screening of hybridoma supernatants. We show that this approach is not only facile but general: it is not limited by type of reaction, substrate, or catalyst (enzymes, catalytic antibodies, chemical catalysts). catELISA opens a route to catalytic antibodies that replaces existing lengthy and arduous methods, thus allowing us to expand their number and improve their quality and to address questions that would otherwise be difficult to answer.
Animals↗
Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the immunoglobulin and T-cell receptors.
The generation of tumor-specific lymphocytes and their use in adoptive immunotherapy is limited to a few malignancies because most spontaneous tumors are very weak or not at all immunogenic. On the other hand, many anti-tumor antibodies have been described which bind tumor-associated antigens shared among tumors of the same histology. Combining the variable regions (Fv) of an antibody with the constant regions of the T-cell receptor (TCR) chains results in chimeric genes endowing T lymphocytes with antibody-type specificity, potentially allowing cellular adoptive immunotherapy against types of tumors not previously possible. To generalize and extend this approach to additional lymphocyte-activating molecules, we designed and constructed chimeric genes composed of a single-chain Fv domain (scFv) of an antibody linked with gamma or zeta chains, the common signal-transducing subunits of the immunoglobulin receptor and the TCR. Such chimeric genes containing the Fv region of an anti-trinitophenyl antibody could be expressed as functional surface receptors in a cytolytic T-cell hybridoma. They triggered interleukin 2 secretion upon encountering antigen and mediated non-major-histocompatibility-complex-restricted hapten-specific target cell lysis. Such chimeric receptors can be exploited to provide T cells and other effector lymphocytes, such as natural killer cells, with antibody-type recognition directly coupled to cellular activation.
Animals↗