Construction, expression, and biologic activity of murine/human chimeric antibodies with specificity for the human alpha/beta T cell receptor.

Murine/human chimeric antibodies with specificity for the human TCR-alpha/beta have been produced by genetic engineering. The L and H chain V region exons encoding the murine mAb BMA 031 were isolated and inserted into mammalian expression vectors containing the human kappa and gamma 1 or gamma 4 C region exons. The chimeric genes were transfected into murine Sp2/O hybridoma cells by electroporation and transfectomas secreting chimeric antibody were isolated. Secretion levels ranged from 1 to 7 pg/cell/24 h. The chimeric antibodies bound specifically to T cells and competed effectively with the parental murine mAb for binding to these sites. The ability to promote antibody-dependent cell-mediated cytolysis was significantly enhanced in the chimeric antibodies as compared with murine BMA 031. C-dependent cytolysis, however, was not detectable with any of the antibodies. Chimeric BMA 031 is a clinically relevant, genetically engineered antibody with potential uses in transplantation, graft-vs-host disease, autoimmune diseases and other T cell-related disorders.     

Characterization and expression of the human alpha beta T cell receptor by using a framework monoclonal antibody

A monoclonal antibody (mAb) with framework reactivity against the T cell receptor (TCR) alpha beta complex is characterized. The mAb, beta Framework 1 (beta F1) is capable of immunoprecipitating the TCR alpha beta complex from 125I-labeled human T cell tumors, immunocompetent T cell clones, and peripheral blood lymphocytes (PBL). beta F1 recognizes the separated TCR beta subunit in Western blotting. Because it does not bind to the surface of viable T cells but does react with the plasma membrane form of the TCR after treatment with membrane solubilizing agents, the beta F1 mAb reacts with a "hidden" determinant on the TCR beta subunit. After solubilization with 70% ethanol, the TCR alpha beta complex is shown to exist on greater than 92% of T3+ human PBL, whereas 2 to 8% of T3+ PBL do not react with the mAb. The beta F1 mAb demonstrates the existence of differently glycosylated surface 125I-labeled TCR alpha-chains (alpha, alpha', alpha") in association with a common TCR beta-chain on the HPB-MLT T cell leukemia. Reactivity of the beta F1 mAb on thymus tissue sections is similar to that of anti-Leu-4 (anti-T3). The beta F1 mAb should prove useful as a research tool for both the immunochemical characterization and isolation of virtually any alpha beta T cell receptor, whether from individual T cell clones or polyclonal populations of T lymphocytes. Recognition of T cell receptors in histologic tissue sections suggests that the beta F1 mAb may be useful in the clinical diagnosis of T cell lineage neoplasms. In failing to recognize all T3+ lymphocytes, it allows the identification of novel populations of T3+ lymphocytes that may express non-alpha, non-beta T cell receptors.     

Assembly, intracellular processing, and expression at the cell surface of the human alpha beta T cell receptor/CD3 complex. Function of the CD3-zeta chain

The TCR/CD3 complex is a multimeric protein complex composed of a minimum of seven transmembrane chains (TCR alpha beta-CD3 gamma delta epsilon zeta 2). Whereas earlier studies have demonstrated that both the TCR-alpha and -beta chains are required for the cell surface expression of the TCR/CD3 complex, the role of the CD3 chains for the TCR/CD3 expression have not been experimentally addressed in human T cells. In this study the function of the CD3-zeta chain for the assembly, intracellular processing, and expression of the TCR/CD3 complex in the human leukemic T cell line Jurkat was investigated. The results indicate that: 1) CD3-zeta is required for the cell surface expression of the TCR/CD3 complex; 2) the pentameric form (TCR alpha beta-CD3 gamma delta epsilon) of the TCR/CD3 complex and single TCR chains associated with CD3 (TCR alpha-CD3 gamma delta epsilon and TCR beta-CD3 gamma delta epsilon) are produced in the endoplasmic reticulum in the absence of CD3-zeta; 3) the CD3-zeta does not associate with TCR alpha-CD3 gamma delta epsilon or TCR beta-CD3 gamma delta epsilon complexes; 4) CD3-zeta associate with the pentameric form of the TCR/CD3 complex in the endoplasmic reticulum to form the heptameric complex (TCR alpha beta-CD3 gamma delta epsilon----TCR alpha beta-CD3 gamma delta epsilon 2); and 5) CD3-zeta is required for the export of the TCR/CD3 complex from the endoplasmic reticulum to the Golgi apparatus for subsequent processing.     

Exclusion and inclusion of alpha and beta T cell receptor alleles.

Exclusion and inclusion of T cell receptor (TCR) genes were analyzed in alpha beta TCR transgenic mice. Both transgenes are expressed unusually early on the surface of CD4-8-, HSA+, IL-2R- thymocytes. These progenitor cells give rise to progeny, which at the single-cell level contains endogenous alpha but not beta TCR-RNA as well as protein, in addition to products encoded by the transgenes. Thus, the surface expression of an alpha beta TCR does not prevent further alpha TCR rearrangement in immature thymocytes that still transcribe RAG-1 and RAG-2 genes. Reduced levels of RAG-1 and RAG-2 RNA are detectable only in CD4+8+ TCR high cells, which result from positive selection in the thymus. The results suggest that a developing T cell may try different alpha beta TCRs for binding to thymic MHC ligands, and that recombination at the alpha locus ceases only after positive selection.     

Alpha beta lineage-specific expression of the alpha T cell receptor gene by nearby silencers

T cells expressing either the alpha beta or gamma delta antigen receptor (TCR) are distinct cell lineages. The single locus encoding the TCR alpha and delta genes requires special regulation to avoid alpha gene expression in gamma delta T cells. We show here that the minimal alpha enhancer is active in the gamma delta T cell lineage but gains alpha beta lineage specificity through negative cis-acting elements 3' of the C alpha gene that silence the enhancer in gamma delta T cells. The negative elements at the C alpha locus consist of several silencers that work in an orientation- and distance-independent fashion. These silencers also act on a retroviral enhancer that is normally ubiquitously expressed, restricting its activity to alpha beta cells. The alpha silencers are active in non-T cell lines, suggesting that the decision of a cell to differentiate into the alpha beta T cell lineage may involve specific relief from these silencers. Silencers are likely to be as important as enhancers in establishing lineage-specific gene expression in many systems.     

Construction and expression of humanized chimeric T cell receptor specific for chronic myeloid leukemia cells

Chimeric T cell receptors (chTCRs), composed of the single-chain variable fragments (scFv) of murine antibodies and human signaling molecules, are used to redirect the specificity of autologous or allogeneic T lymphocytes. To develop novel therapeutic agents for treatment of chronic myeloid leukemia (CML), we engineered a scFv from the hybridoma cell line CMA1 which produces monoclonal antibody specific against CML. The genes encoding the heavy and light chain variable regions were amplified from CMA1 cDNA and a humanized chTCR was constructed. Expression of the novel hchTCR was verified in NIH3T3 cells transduced with retroviral vectors. The results demonstrated that hchTCR can be expressed and presented on cell surface normally. These results suggest that retroviral vectors expressing hchTCR specific for CML cells may be used to redirect human T lymphocytes.     

Monoclonal antibodies directed against the human A431 beta 2-adrenergic receptor recognize two major polypeptide chains

A serum-albumin-alprenolol conjugate was used to isolate beta-adrenergic receptors from the human A431 cell lysates. Three monoclonal antibodies were obtained from BALB/c mice immunized with these receptors. These antibodies: BRK-1, BRK-2, BRK-3, were respectively of the IgM, IgG2a and IgG3 classes. All three antibodies recognized photoaffinity-labelled receptors, immunoprecipitated ligand-binding activity and identified the 65-kDa and 55-kDa polypeptides corresponding to the beta 2-adrenergic receptors of A431 cells. BRK-2 and BRK-3 recognized both beta 1 and beta 2-adrenergic receptors of several mammalian cells. All three antibodies visualized, by immunofluorescence, the beta 2-adrenergic receptors at the surface of A431 cells. The monoclonal antibodies are directed against the protein portion of the beta-adrenergic receptors since partial or complete removal of the carbohydrate moieties by treatment with endoglycosidase such as endo-F (endo-beta-N-acetylglucosaminidase F) and periodate oxidation did not affect the immunoreactivity. These antibodies will be of value to immunopurify the beta-adrenergic receptors.     

Site-specific antibodies directed against G protein beta and gamma subunits: effects on alpha and beta gamma subunit interaction.

Little is known about the specific domains of G protein beta and gamma subunits which interact with each other and with the alpha subunit. We used site-specific anti-peptide antibodies directed against beta and gamma subunits to investigate domains on beta and gamma subunits involved in alpha subunit interaction. Antibodies included four against the transducin (Gt) beta subunit (residues 1-10 = MS, 127-136 = KT, 256-265 = RA, and 330-340 = SW) and two against the gamma subunit (residues 2-12 = PV and 58-68 = PE). All antisera, when affinity-purified on peptide columns, yielded antibodies capable of recognizing the denatured cognate subunit on immunoblots, but only RA, SW, PV, and PE recognized native beta gamma t subunits. Affinity purification of MS and KT antisera on columns of immobilized native Gt yielded antibodies capable of recognizing native beta gamma t subunits. The functional effects of each antibody preparation on alpha t-beta gamma t interaction were assessed by assaying the ability of the preparations to immunoprecipitate beta gamma t subunits in the presence of excess alpha subunits and by testing the inhibition of beta gamma t-dependent ADP-ribosylation of alpha t-subunits catalyzed by pertussis toxin. On the basis of the results, we conclude that the domains on beta gamma t which may be directly involved in alpha t-beta gamma t interaction include the extreme amino terminus, residues 127-136 and 256-265 of beta t, and the carboxyl terminus of gamma t.(ABSTRACT TRUNCATED AT 250 WORDS)     

Marked differences in the efficiency of expression of distinct alpha beta T cell receptor heterodimers

Ag recognition by most T lymphocytes is mediated by clonally distributed alpha beta heterodimeric receptors. A major fraction of TCR diversity is believed to be due to the random coexpression in individual T cells of the products of independently rearranging alpha- and beta-genes (combinatorial diversity). However, analysis of cell surface receptors on transfected T hybridoma cells synthesizing various sets of alpha- and beta-chains revealed marked differences in the efficiency of expression of certain alpha beta-pairs. Specifically, using the functionally rearranged gene products of the 2B4 cytochrome c specific T hybridoma (V beta 3, V alpha 11.2) and BW5147 parent lymphoma (V beta 1, V alpha BW), a hierarchy of expression efficiency relative to indirectly measured precursor chain levels in the cell was shown to be 2B4 alpha-BW beta greater than 2B4 alpha - 2B4 beta greater than BW alpha - BW beta greater than BW alpha - 2B4 beta. The estimated difference between the best expressed and worst expressed pairs is on the order of 50-fold. For the beta-chain, the primary determinant of expression efficiency with a given alpha-chain appears to be the V segment, as a second V beta 1-chain with distinct D and J regions from BW beta was expressed with the same pattern. These data imply that alpha- and beta-chains do not form well-expressed TCR in a random manner and that limitations on the useful combinatorial association of these chains may significantly affect the functional T cell repertoire.     

Construction and characterization of functional anti-epiregulin humanized monoclonal antibodies

Growth factors are implicated in several processes essential for cancer progression. Specifically, epidermal growth factor (EGF) family members, including epiregulin (EREG), are important prognostic factors in many epithelial cancers, and treatments targeting these molecules have recently become available. Here, we constructed and expressed humanized anti-EREG antibodies by variable domain resurfacing based on the three-dimensional (3D) structure of the Fv fragment. However, the initial humanized antibody (HM0) had significantly decreased antigen-binding affinity. Molecular modeling results suggested that framework region (FR) residues latently important to antigen binding included residue 49 of the light chain variable region (VL). Back mutation of the VL49 residue (tyrosine to histidine) generated the humanized version HM1, which completely restored the binding affinity of its murine counterpart. Importantly, only one mutation in the framework may be necessary to recover the binding capability of a humanized antibody. Our data support that HM1 exerts potent antibody-dependent cellular cytotoxicity (ADCC). Hence, this antibody may have potential for further development as a candidate therapeutic agent and research tool.     

Suppression of chronic antigen-induced arthritis in rats by a monoclonal antibody against the T cell receptor alpha beta.

We have investigated a role for T cells in chronic antigen-induced arthritis in rats employing a monoclonal antibody (R73 mAb) against the T cell receptor alpha beta. Treatment with R73 mAb from the time of intra-articular antigenic challenge blocked completely the induction of chronic, but not acute ovalbumin-induced arthritis in sensitized rats. Histologically, treatment-controlled arthritic rats exhibited marked hyperplasia of synovial membrane with pronounced infiltration of inflammatory cells including alpha beta + T cells in the chronic phase of arthritis. In contrast, R73 mAb-treated rats had almost normal joint histology. Treatment with R73 mAb after onset of arthritis was also effective in suppressing the progression of chronic antigen-induced joint inflammation. The preventive and suppressive effects of the mAb on chronic antigen-induced arthritis were associated with marked depletion of alpha beta + T cells in peripheral blood. The DTH but not the humoral response to ovalbumin in sensitized rats was suppressed significantly by R73 mAb. Thus, alpha beta + T cells appear to have a central role in both induction and progression of chronic antigen-induced arthritis.     

Potentiation of transmembrane signaling by cross-linking of antibodies against the beta chain of the T cell antigen receptor of JURKAT T cells.

Three monoclonal antibodies (mAb) 2D1, 3B9, and 3B12 were produced by immunizing BALB/c mice with JURKAT cells. These mAb induce comodulation of the TCR/CD3 complex expressed on JURKAT cells, but do not react with the CD3- JURKAT variant, J.RT3.T3.1. Immunoprecipitation studies with detergent-solubilized JURKAT cell lystes indicate that these mAb react with proteins having characteristics of the TCR molecules. Their low reactivity with peripheral blood mononuclear cells (PBMC) and lack of reactivity with other CD3+ T cell lines suggest that they may be anti-idiotypic mAb. Results from binding inhibition assays, reactivity with PBMC, and generation of transmembrane signals suggest that these three anti-TCR mAb recognized different epitopes on the TCR beta chain of JURKAT cells. Although the three mAb are capable of inducing the production of inositol phosphates and cytosolic free Ca2+ increase in JURKAT cells, their stimulatory capacities vary and are lower than that observed by anti-CD3 antibody (OKT3) stimulation. However, crosslinking these mAb with rabbit antimouse immunoglobulins potentiates the stimulatory response to comparable levels induced by OKT3. These mAb could be useful as tools to study V beta 8+ T cells in relation to antigen-specific activation.     

Production and characterization of a humanized single-chain antibody against human integrin alphav beta3 protein

Anti-angiogenesis therapy is an emerging strategy for cancer treatment. This therapy has many advantages over existing treatments, such as fewer side effects, fewer resistance problems, and a broader tumor type spectrum. Integrin αvβ3 is a heterodimeric transmembrane glycoprotein that has been demonstrated to play a key role in tumor angiogenesis and metastasis. We have used a phage antibody display to humanize a mouse monoclonal antibody (mAb E10) against human integrin αvβ3 with a predetermined CDR3 gene. Three human phage antibodies were developed. Analysis of the humanized phage antibodies by phage ELISA revealed that the antibodies retained high antigen-binding activity and detected the same epitope as the parent mAb E10. A humanized single chain Fv (scFv) antibody was expressed in Escherichia coli in a soluble form. Analysis of the purified scFv indicated that it has the same specificity and affinity as the original mAb. Cell viability assays and xenograft model results suggested that the humanized scFv possesses anti-tumor growth activity in vitro and in vivo. This successful production of a humanized scFv with the ability to inhibit αvβ3-mediated cancer cell growth may provide a novel candidate for integrin αvβ3-targeted therapy.     

Random length assortment of human and mouse T cell receptor for antigen alpha and beta chain CDR3.

In view of the recently determined three-dimensional structures of complexes formed by the T cell receptor for antigen (TCR), the processed peptide and the MHC class I molecule, it is expected that the combined configuration formed by the third complementarity determining regions (CDR3) of TCR alpha and beta chains will be very restricted in size and shape due to the limited length variations of the processed peptides. Thus, the combined TCR alpha and beta chain CDR3 lengths should have a fairly narrow distribution. This feature can be due to the selective association of long alpha chain CDR3 with short beta chain CDR3 and vice versa or due to random assortment of alpha and beta chain CDR3 of even narrower length distribution. Based on existing translated amino acid sequence data, it has been found that the latter mechanism is responsible.     

Production and isolation of rabbit anti-idiotypic antibodies directed against the human monocyte receptor for yeast beta-glucans.

beta-Glucan receptors are present on mammalian phagocytic cells and provide an important physiologic mechanism for opsonin-independent clearance of yeasts and fungi. To prepare an immunologic probe to human monocyte beta-glucan receptors, an approach was taken that focused on the ligand specificity of the receptors as expressed by an anti-Id. The algal beta-glucan laminarin was chemically coupled to protein carriers to prepare an immunogenic beta-glucan. Three mouse IgG2a mAb were raised against laminarin, and one, mAb OEA10, exhibited specificity for the soluble unit ligand yeast heptaglucoside and the ligands present on zymosan and glucan particles that are recognized by monocyte beta-glucan receptors. These findings prompted usage of mAb OEA10 as immunogen for the preparation of an anti-Id. The resulting rabbit antiserum was subjected to sequential immunoaffinity chromatography to purify anti-idiotypic antibodies. The final product contained only IgG by SDS-PAGE and was shown to be specific by its selectively blocking binding of 125I-mAb OEA10 to laminarin. Pretreatment of adherent monocytes with 0.4 micrograms/ml of the anti-Id reduced the numbers of monocytes ingesting zymosan and glucan particles by 64 and 43%, respectively, whereas ingestion of IgG-coated SRBC was unaffected by as much as 16 micrograms/ml of the anti-Id. Incubation of adherent monocytes with increasing amounts of 125I-anti-Id in the absence and presence of 40-fold molar excess unlabeled anti-Id revealed dose-dependent specific binding, which approached plateau levels with 1 microgram/ml of labeled anti-Id. Thus, the anti-Id binds to monocytes and displays functional characteristics of soluble beta-glucan ligands, indicating that some of the anti-idiotypic antibodies recognize epitopes within monocyte beta-glucan receptors.     

Phenotype, ontogeny, and repertoire of CD4-CD8- T cell receptor alpha beta + thymocytes. Variable influence of self-antigens on T cell receptor V beta usage

We have characterized CD4-CD8- double negative (DN) thymocytes that express TCR-alpha beta and represent a minor thymocyte subpopulation expressing a markedly skewed TCR repertoire. We found that DN TCR-alpha beta + thymocytes resemble mature T cells in that they (a) are phenotypically CD2hiCD5hiQa2+HSA-, (b) appear late in ontogeny, and (c) are susceptible to cyclosporin A-induced maturation arrest. In addition, we found that DNA sequences 5' to the CD8 alpha gene were demethylated relative to their germline state, suggesting that DN TCR-alpha beta + thymocytes are derived from cells that had at one time expressed their CD8 alpha gene locus. Because DN TCR-alpha beta + thymocytes are known to express an unusual TCR repertoire with significant overexpression of V beta 8, we were interested in examining the possible role played by self-Ag in shaping their TCR repertoire. It has been suggested that DN TCR-alpha beta + thymocytes are derived from potentially self-reactive thymocytes that have escaped clonal deletion by down-regulating their surface expression of CD4 and/or CD8 determinants. However, apparently inconsistent with such an hypothesis, we found that the frequency of DN thymocytes expressing various anti-self TCR (V beta 6, V beta 8.1, V beta 11, V beta 17a) were not increased in strains expressing their putative self-Ag, but instead were either unaffected or significantly reduced in those strains. With regard to V beta 8 expression among DN TCR-alpha beta + thymocytes, V beta 8 overexpression in DN TCR-alpha beta + thymocytes appeared to be independent of, and superimposed on, the developmental appearance of the basic DN thymocyte repertoire. Even though V beta 8 overexpression appeared to be generated by a mechanism distinct from that generating the rest of the DN TCR-alpha beta + thymocyte repertoire, we found that super-Ag against which V beta 8 TCR react introduced into the neonatal differentiation environment also significantly reduced, rather than increased, the frequency of DN TCR-alpha beta + V beta 8+ thymocytes. Thus, the present study is consistent with DN TCR-alpha beta + thymocytes being mature cells derived from CD8+ precursors, and documents that their TCR repertoire can be influenced, at least negatively, by either self-Ag or Ag introduced into the neonatal differentiation environment. However, we found no evidence to support the hypothesis that DN TCR-alpha beta + thymocytes are enriched in cells expressing TCR reactive against self-Ag.