Purification of an active EGF receptor kinase with monoclonal antireceptor antibodies

A method is described for a rapid two-step purification of the membrane receptor for epidermal growth factor (EGF) from cultured human A-431 cells. After solubilization of the cells with Triton X-100, the receptor is immobilized on an immunoaffinity column containing a monoclonal antibody directed against the receptor. In the second step of purification, the receptor, eluted from the antibody column, is adsorbed and specifically eluted from a lectin-agarose column. The molecular species obtained is mainly the 170,000-dalton EGF receptor polypeptide. The activity of the pure receptor depends on the conditions used for the desorption from the immunoaffinity beads. High-yield elution is obtained with acidic buffer and the receptor so purified specifically binds EGF, but is devoid of the kinase activity. When the elution is done with alkaline buffers or with buffer containing urea, a fully active receptor kinase is purified (yield of 10%). The pure receptor binds 125I-EGF with a Kd of 4 X 10(-8) M and retains EGF-sensitive protein kinase activity which phosphorylates tyrosine residues on the receptor itself. An additional protocol is described for large-scale purification (yield of 55%) of EGF receptor for the analysis of its primary structure. In this procedure, the EGF receptor is first purified by immunoaffinity chromatography which is followed by preparative gel electrophoresis of the 32P internally labeled receptor to remove minor protein contaminants.     

Analysis of the anti-alpha 1 leads to 3 dextran response with monoclonal anti-idiotype antibodies

The antibody response to alpha 1 leads to 3 dextran (DEX) in BALB/c mice consists of a family of closely related yet highly heterogeneous molecules. Although these antibodies have been previously characterized both idiotypically and structurally, detailed analysis of responding clones has not been possible using conventional anti-idiotype antibodies. Monoclonal syngeneic and allogeneic anti-idiotype antibodies (MAIDs) specific for anti-DEX antibodies were used in this study to dissect the serum antibody response to DEX in BALB/c mice. The constructed MAIDs showed considerable heterogeneity by isoelectric focusing and by their binding characteristics to a series of DEX specific myeloma and hybridoma proteins. The predominant heavy chain isotype of these MAIDs was gamma 1. These antibodies were used to identify individual idiotypic structures (IdI) on J558, or M104E as well as cross-reactive determinants common to both (IdX). Although both IdX and IdI MAIDs were obtained, IdI specific antibodies were obtained more frequently. BALB/c mice immunized with DEX produced antibodies expressing both IdI but in highly variable amounts. A large percentage of, but not all DEX specific antibody, could be accounted for by IdX bearing antibodies. Suppression of adult and neonatal mice by IdI specific MAIDs was effective with precise elimination of only those clones expressing IdI determinants leaving the total lambda bearing anti-DEX response intact. Suppression of adults and neonates by an IdX specific MAID resulted in a temporary and partial suppression of the total lambda bearing anti-DEX response along with total suppression of the IdX portion of the response. Unlike other systems these monoclonal antibodies produce only suppression, and under a variety of conditions enhancement of anti-DEX responses has not been observed.     

Interaction of murine progesterone receptors with specific monoclonal antibodies to the avian progesterone receptor

Monoclonal antibodies raised against purified chicken progesterone receptor (PgR) have been described and characterized recently. In this study we have screened these antibodies for cross-reactivity with murine PgR. Of the six anti-PgR antibodies tested, one (alpha PR6) precipitates murine PgR in an assay using protein A-sepharose as an absorbent for the antibody. The antibody is specific for PgR and does not react with the estrogen receptor or the glucocorticoid receptor in the same cytosol. In immunoblot experiments, both alpha PR6 and alpha PR11 recognize a 115,000 Da protein, however, alpha PR11 gives a weaker signal than alpha PR6. In photoaffinity labeling experiments, a 115,000 Da and an 83,000 Da protein covalently bind tritiated R5020 in a receptor-specific way. We conclude that the alpha PR6 antibody can be used as a tool to study the structure and function of the murine PgR.     

Heterogeneity of growth-hormone receptors detected with monoclonal antibodies to human growth hormone.

The specificity of hormone-receptor interactions has been examined with the aid of monoclonal antibodies (MABs) (EB1, EB2, QA68 and NA71) defining four non-overlapping antigenic determinants on human growth hormone (hGH). The results indicate that growth-hormone receptors in liver obtained from different sources differ with regard to their affinities and relative numbers; they may also differ with respect to the region of the growth-hormone molecule to which they bind. Antibody NA71 effectively inhibited hormone binding to all receptor preparations tested, although with various degrees of potency. Monoclonal antibody EB1 demonstrated a graded inhibition with respect to its ability to block 125I-hGH binding to receptors from various sources, the maximum inhibition being seen in receptor preparations from mouse and ovine liver and the minimum in rat liver. MABs EB2 and QA68 also showed various abilities to inhibit hormone-receptor interaction, depending on the origin of the receptor preparation. Furthermore, the receptor-binding characteristics of hormone-antibody complexes were dependent on whether the binding-site preparation was derived from pregnant, lactating or 'normal' animals. A particularly striking difference between the ability of hormone-MAB complexes to bind to receptors from different sources was seen for microsomes (microsomal fractions) derived from livers of animals of the 'Little' mouse strain. These animals become progressively obese, and it was shown that MABs were considerably more effective in inhibiting 125I-labelled hGH binding to microsomes from phenotypically obese mice than to those derived from their non-obese littermates. The results can be explained by the presence of multiple receptor types for GH, the relative proportions of which vary according to the physiological state of the animal, and possibly between species.     

Intracellular forms of Drosophila topoisomerase II detected with monoclonal antibodies.

We developed monoclonal antibodies against Drosophila topoisomerase II and studied the intracellular forms and the in vivo and in vitro proteolytic degradation of the enzyme. In purified enzyme preparations polyclonal sera and monoclonal antibodies recognized several polypeptides in the 170-132 kD molecular weight range. In vivo, however, the pattern was much simpler. In Drosophila embryos, pupae, fly heads and Schneider S3 tissue culture cells topoisomerase II appeared as a single 166 kD polypeptide. In Drosophila embryos, with two monoclonal antibodies topoisomerase II appeared as a doublet composed of the 166 kD canonical form and a slightly higher molecular weight polypeptide. Topoisomerase II was shown to be present also in fly heads which are composed entirely of nonproliferative tissues.     

Endocytosis, recycling, and degradation of the insulin receptor. Studies with monoclonal antireceptor antibodies that do not activate receptor kinase

The endocytosis, recycling, and degradation of the insulin receptor were studied in IM-9 cells and U-937 cells by employing two monoclonal antibodies directed at the alpha subunit of the human insulin receptor, antibodies MA-5 and MA-10. Antibody MA-5 is an insulin agonist and MA-10 is an insulin antagonist (Forsayeth, J., Caro, J.F., Sinha, M.K., Maddux, B.A., and Goldfine, I.D. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 3448-3451). Both monoclonal antibodies, like insulin, induced the endocytosis of the insulin receptor within 15 min. Upon removal of extracellular ligand the internalized receptor recycled to the cell surface. At this time there was no degradation of the receptor as measured by a sensitive insulin receptor radioimmunoassay. After 20 h of incubation, insulin and MA-5, but not MA-10, induced significant receptor degradation as measured by both insulin receptor radioimmunoassay and metabolic labeling studies. These studies demonstrated, therefore, that: 1) internalization and recycling of the receptor can be induced by antireceptor monoclonal antibodies that are either insulin agonists or insulin antagonists; 2) enhanced receptor degradation can be induced by monoclonal antibodies that are insulin agonists; and 3) the process of receptor internalization does not necessarily lead to enhanced receptor degradation. Since prior studies have indicated that neither MA-5 nor MA-10 enhance insulin receptor kinase activity, the present studies also suggest that insulin receptor endocytosis and degradation induced by ligands different than insulin can occur without activation of this process.     

Modulation of two forms of tumor necrosis factor receptors and their cellular response by soluble receptors and their monoclonal antibodies.

Recently, two different receptors for human tumor necrosis factor (TNF) with molecular masses of 60 kDa (p60) and 80 kDa (p80) have been identified. In this report, we investigated the effect of the soluble forms of these receptors and monoclonal antibodies against them on ligand interaction, receptor down-regulation, and mediation of cellular response in U-937 cells. Our results indicate that p60 and p80 constitute 20-30 and 60-80% of the total TNF-binding sites on U-937 cells, respectively. However, by cross-linking, only the p80 form of the receptor could be detected. In contrast to unlabeled TNF, the anti-p60 and anti-p80 antibodies together only partially inhibited ligand binding, and this inhibition was not additive. Lack of additive inhibition of binding was found to be not due to stereo-chemical hindrance. TNF binding to cells can be completely displaced by soluble forms of either the p60 or p80 receptor. However, 100-fold more of the p80 than the p60 form of the soluble receptor is needed for equivalent displacement. Under optimum conditions, TNF and the anti-p80 and anti-p60 antibodies down-regulated 30, 80, and 20% of the TNF receptors, respectively. The anti-p60 and anti-p80 antibodies down-regulated not only their own receptors, but also reciprocal receptors, suggesting a cross-communication between the p60 and p80 forms of the TNF receptor. In spite of inhibiting as much as 80% of TNF binding, none of the receptor antibodies significantly inhibited the cytotoxic response to TNF in U-937 cells. Soluble forms of both receptors, however, completely abrogated the cellular response to TNF. Thus, overall, our results indicate that the antibodies against both receptors together inhibit the majority of the receptor-ligand interaction without any significant effect on the biological response to TNF.     

Functional analysis of TRAIL receptors using monoclonal antibodies

mAbs were generated against the extracellular domain of the four known TNF-related apoptosis-inducing ligand (TRAIL) receptors and tested on a panel of human melanoma cell lines. The specificity of the mAb permitted a precise evaluation of the TRAIL receptors that induce apoptosis (TRAIL-R1 and -R2) compared with the TRAIL receptors that potentially regulate TRAIL-mediated apoptosis (TRAIL-R3 and -R4). Immobilized anti-TRAIL-R1 or -R2 mAbs were cytotoxic to TRAIL-sensitive tumor cells, whereas tumor cells resistant to recombinant TRAIL were also resistant to these mAbs and only became sensitive when cultured with actinomycin D. The anti-TRAIL-R1 and -R2 mAb-induced death was characterized by the activation of intracellular caspases, which could be blocked by carbobenzyloxy-Val-Ala-Asp (OMe) fluoromethyl ketone (zVAD-fmk) and carbobenzyloxy-Ile-Glu(OMe)-Thr-Asp (OMe) fluoromethyl ketone (zIETD-fmk). When used in solution, one of the anti-TRAIL-R2 mAbs was capable of blocking leucine zipper-human TRAIL binding to TRAIL-R2-expressing cells and prevented TRAIL-induced death of these cells, whereas two of the anti-TRAIL-R1 mAbs could inhibit leucine zipper-human TRAIL binding to TRAIL-R1:Fc. Furthermore, use of the blocking anti-TRAIL-R2 mAb allowed us to demonstrate that the signals transduced through either TRAIL-R1 or TRAIL-R2 were necessary and sufficient to mediate cell death. In contrast, the expression of TRAIL-R3 or TRAIL-R4 did not appear to be a significant factor in determining the resistance or sensitivity of these tumor target cells to the effects of TRAIL.     

Dissociation of alloantigen recognition from self major histocompatibility complex-restricted recognition of cytolytic T lymphocytes by monoclonal antireceptor antibodies

Two monoclonal antibodies (mAb) directed to the dual reactive cytolytic T lymphocyte clone OH8 (Db + H-Y and H-2d) were established. Analysis by cell surface staining and immunoprecipitation of radiolabeled surface molecules of OH8 followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that both mAb recognized an identical heterodimeric, clonotypic structure on OH8 cells, i.e., T cell receptor. However, although the MR3-2 mAb inhibited the lysis of either Db + H-Y or H-2d targets by OH8, the MR3-6 mAb inhibited the lysis of H-2d target cells, but not that of Db + H-Y target cells. Modulation of T cell receptor by either MR3-2 or MR3-6 mAb rendered the OH8 cytolytic T lymphocyte incapable of killing both Db + H-Y and H-2d target cells. These findings suggest that different epitopes of OH8 T cell receptor were involved for the recognition of self + antigen and alloantigen.     

Interaction of antibodies with ErbB receptor extracellular regions

Antibodies to the extracellular region of the ErbB receptors have played key roles in the development of a mechanistic understanding of this family of receptor tyrosine kinases. An extensively studied class of such antibodies inhibits activation of ErbB receptors, and these antibodies have been the focus of intense development as anti-cancer agents. In this review we consider the properties of ErbB receptors antibodies in light of the current structure-based model for ErbB receptor homo- and hetero-dimerization and activation. Crystal structures of the Fab fragments from five different inhibitory antibodies in complex with the extracellular regions of EGFR and ErbB2 have been determined. These structures highlight several different modes of binding and mechanisms of receptor inhibition. Information about antibody interactions with the structurally well-characterized soluble extracellular regions of ErbB receptors can be combined with the rich knowledge of the effects of these antibodies in cultured cells, and in vivo, to provide insights into the conformation and activation of ErbB receptors at the cell surface.     

Targeted disruption of the ABP-120 gene leads to cells with altered motility

The actin-binding protein ABP-120 has been proposed to play a role in cross-linking F-actin filaments during pseudopod formation in motile Dictyostelium amebas. We have tested this hypothesis by analyzing the phenotype of mutant cell lines which do not produce ABP-120. Two different transformation vectors capable of targeted disruption of the ABP-120 gene locus have been constructed using a portion of an ABP-120 cDNA clone. Three independent cell lines with different disruption events have been obtained after transformation of amebas with these vectors. The disruption of the ABP-120 gene by vector sequences results in either the production of a small amount of truncated ABP-120 or no detectable protein at all. The phenotypes of two different clones lacking ABP-120, generated in strains AX3 and AX4, have been characterized and show identical results. ABP-120- cells tend to remain rounder before and after cAMP stimulation, and do not reextend pseudopods normally after rapid addition of cAMP. In addition, ABP-120- cells translocating in buffer exhibit defects in both the rate and extent of pseudopod formation. The amount of F-actin cross-linked into the cytoskeleton after cAMP stimulation of ABP-120- cells is reduced at times when ABP-120 has been shown to be incorporated into the cytoskeleton, and this correlates temporally with the absence of reextension of pseudopods after cAMP stimulation. The instantaneous velocity is significantly reduced both before and after cAMP stimulation in the ABP-120- cells, and the cells show decreased chemotactic efficiency compared to ABP-120+ controls. This phenotype is consistent with a role for ABP-120 in pseudopod extension by cross-linking actin filaments as proposed by the "cortical expansion model" (Condeelis, J., A. Bresnick, M. Demma, C. Dharmawardhane, R. Eddy, A. L. Hall, R. Sauterer, and V. Warren. 1990. Dev. Genet. 11:333-340).     

The role of antireceptor antibodies in stimulating phosphorylation of the insulin receptor

Four polyclonal antisera directed against the insulin receptor were tested for their capability to activate the tyrosine-specific protein kinase associated with the receptor. All four antisera were shown to inhibit insulin binding to the receptor in cultured human lymphoblastoid cells and to stimulate lipogenesis in isolated rat adipocytes. Although two antisera (B-d, B-8) stimulated the activity of the tyrosine kinase of partially purified receptor preparations from rat liver, two other antisera (B-2 and B-10) failed to do so. This failure could not be explained by lack of antibody binding to receptor, by interference with the receptor as a substrate for the kinase, or by blocking of the enzyme's active site. We conclude that these two antireceptor antibodies bind to the receptor but fail to activate the kinase. The simplest interpretation of these observations is that activation of the tyrosine-specific protein kinase might not be an obligatory step in coupling insulin binding to insulin action. However, it is also possible that the mechanism by which polyclonal antireceptor antisera mimic insulin's bioactivity may differ from the mechanism of action of insulin itself.     

Pathophysiology of estrogen receptors in mammary tissue by monoclonal antibodies

Identification of preneoplastic lesions of the breast has mainly rested on morphological grounds, supported by epidemiological data. These studies assign a definite precancerous potential to a group of atypical hyperplastic lesions and in situ carcinoma. In spite of much effort no criteria are yet available to understand which, among these lesions, is committed to infiltrative growth, in other words, to understand the risk to a single patient. Estrogens are know to play a critical role in the etiology of breast cancer. The hypothesis is investigated that this role is dependent on a modified expression of their receptor. To approach this question estrogen receptor expression was traced by specific monoclonal anti-receptor antibodies and immunocytochemistry, on a spectrum of breast tissue changes, from normal tissue to infiltrating cancer. Estrogen receptor expression is heterogeneous in normal tissue and in infiltrating cancer, and on the contrary is homogeneous in proliferative atypical lesions and in in situ carcinomas. Present results show that receptor expression is enhanced and becomes homogeneous, maybe constitutive, in atypical hyperplasia and in in situ carcinoma and that this phenomenon could subserve important changes of proliferative capacity which are necessary and possibly sufficient for autonomous growth.     

Characterization of monoclonal antibodies directed against trail or trail receptors

A subset of tumour necrosis factor receptor family members is involved in death transducing signals and is, therefore, referred as the "death receptors." Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in many tumour cells but only rarely in normal cells. Five distinct receptors have been described for TRAIL: TRAIL R1 (DR4), TRAIL R2 (DR5, TRICK), TRAIL R3 (TRID, DcR1), TRAIL R4 (TRUNDD, DcR2), and osteoprotegerin. In the Eighth International Workshop on Human Leukocyte Differentiation Antigens, 10 monoclonal antibodies (mAbs) reported to be specific for TRAIL or for TRAIL receptors were submitted. In the present study, the mAb specificity was determined by ELISA. Using these mAbs, investigation on the expression of TRAIL and TRAIL receptors was performed. Some of them were able to modulate TRAIL induced programmed cell death.     

Localization of the site of human transferrin interacting with cellular receptors using monoclonal antibodies]

Antigenic determinants of the human transferrin molecule on the sublobe and lobe levels were localized for 7 monoclonal antibodies. Antibodies used have different effects on the interaction of the transferrin with its receptor. It was concluded that transferrin-receptor recognition was determined by NH2-lobe, the N2-sublobe playing major part. Dimerization of the transferrin molecules in solution was detected. Using the panel of monoclonal antibodies it was shown that dimerization accomplished by means of the COOH-lobes of transferrin molecules, the sites of interaction of the NH2-lobe with receptor being exposed. A model of the transferrin - receptor complex is proposed.