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Query: UNIPROT:P04626 (erbB-2)
 5,251 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bispecific antibodies of a new category, termed "antigen forks", were constructed by crosslinking antibodies that recognized pairs of distinct tumor cell surface antigens. At concentrations of 1-100 nM, several such forks inhibited the growth of human tumor cell lines bearing both relevant antigens. The same cells were not inhibited by unconjugated component antibodies, and the active conjugates did not inhibit the growth of human cell lines that expressed lower levels of relevant antigens. The three most active antigen forks all contained monoclonal antibody 454A12, which recognizes human transferrin receptor. This antibody was conjugated respectively to antibodies 113F1 (against a tumor-associated glycoprotein complex), 317G5 (against a 42-kDa tumor-associated glycoprotein), or 520C9 (against the c-erbB-2 protooncogene product). The 317G5-454A12 fork strongly inhibited the HT-29 and SW948 human colorectal cancer cell lines, while the 113F1-454A12 and 520C9-454A12 forks strongly inhibited the SK-BR-3 human breast cancer cell line and the 113F1-454A12 fork was also effective against SW948. By designing forks against antigens of incompatible function that are co-expressed at high levels on tumor cells but not on normal tissues, it may be possible to generate reagents that inhibit tumor growth with enhanced selectivity.
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PMID:Antigen forks: bispecific reagents that inhibit cell growth by binding selected pairs of tumor antigens. 804 25

Using the breast carcinoma cell line MDA-MB 468 as immunogen, we have produced six new rat monoclonal antibodies (mAbs) against the human EGF receptor (EGFR) and are investigating their use for diagnostic and therapeutic applications in cancer patients whose tumours overexpress these receptors. The mAbs (three IgG2b and one each of IgG2a, IgG1 and IgA) were selected on the basis that they bound to the extracellular domain of the EGFR and blocked growth factor-receptor interaction. Competitive assays showed that, with the exception of antibody ICR65, the mAbs bound to one of two distinct epitopes on the external domain of the EGFR. ICR65, however, cross-reacted with mAbs binding to both epitopes. All of the mAbs immunoprecipitated the 170 kDa glycoprotein from cells expressing the EGFR but not the 185 kDa product of the related c-erbB-2 proto-oncogene. Unlike EGF and TGF alpha none of the mAbs stimulated the growth of quiescent human foreskin fibroblasts but they inhibited the EGF and TGF alpha induced growth stimulation of these cells in vitro. When tested for their effect on tumour cells the mAbs were found to inhibit the growth in vitro of a number of human tumours that overexpressed the EGFR (e.g. HN5, HN6, HN15, A431, MDA-MB 468) but they were without effect on tumour cell lines expressing low or undetectable amounts of the receptor. Our initial results indicate that this new generation of antibodies which bind with high affinity to the EGFR, block growth factor-receptor interaction and inhibit the growth of human squamous carcinoma cell lines overexpressing the receptor have potential for clinical application.
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PMID:The human EGF receptor as a target for cancer therapy: six new rat mAbs against the receptor on the breast carcinoma MDA-MB 468. 809 90

The human c-erbB-2 protooncogene product (erbB-2 protein) is a 185 kilodalton glycoprotein closely related to epidermal growth factor receptor protein. In this study, we measured the concentration of circulating erbB-2 protein in cancer patients by means of a new immunoradiometric assay (IRMA). Two monoclonal antibodies (MoAbs), SV2-61 gamma and 6G10, recognize erbB-2 protein but bind to separate epitopes. SV2-61 gamma was used as an immunoadsorbent and 6G10 as an 125I-labeled probe. A serum was considered positive for erbB-2 protein if the percent binding exceeded the mean of the normal group by more than 3 standard deviations. Eleven of 21 patients with advanced breast cancer and 1 of 15 with advanced gastric cancer were positive. Serum erbB-2 protein levels correlated well with the therapy and the status of the patients with breast cancer. On the contrary, all patients with advanced colon, ovarian, or pancreatic cancers, showed levels below the cut-off value. These results suggest that circulating erbB-2 protein can be measured using the newly constructed IRMA. Since c-erbB-2 protooncogene amplification and overexpression are accepted as a good marker of aggressiveness, relapsing potency, and poor prognosis, this IRMA should be a promising tool with which to help manage breast cancer patients.
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PMID:Construction of immunoradiometric assay for circulating c-erbB-2 protooncogene product in advanced breast cancer patients. 809 2

The oncogene c-erbB-2 has been shown to be amplified in 17-30% of breast cancers, with similar levels of overexpression of the oncogene product p185, a transmembrane growth factor receptor glycoprotein. Amplification of c-erbB-2 is now generally considered to be a significant prognostic indicator in patients with breast cancer. A series of 74 consecutive breast carcinomas were analysed for c-erbB-2 amplification and p185 overexpression. The procedures of Southern blotting and slot blot were used for the analysis of oncogene amplification, while immunoperoxidase (IPOX) staining and enzyme-linked immunosorbent assay (ELISA) were used for the analysis of p185 overexpression. Detection of c-erbB-2 oncogene amplification by both the conventional Southern blotting technique and by the slot blot technique showed complete accord, with the amplified c-erbB-2 oncogene being detected in 14 of the 74 patients (18.9%). The c-erbB-2 oncoprotein, as measured by IPOX and ELISA, was found to be overexpressed in 21% and 19% of patients, respectively. Comparison was made between the results attained by all four methods, and further comparison of the techniques was made from the point of view of ease of use, expense and ease of introduction into routine diagnostic laboratories. Immunocytochemistry in combination with slot blotting procedures were considered to be the most cost effective methods for evaluation of overexpression and amplification in routine pathology laboratories.
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PMID:c-erbB-2 amplification and overexpression in breast cancer: evaluation and comparison of Southern blot, slot blot, ELISA and immunohistochemistry. 810 91

The earliest substrates to the transmembrane insulin receptor tyrosine kinase, that would function in insulin signalling, are likely to be associated with the plasma membrane. Rat liver plasma membrane 180,000 M(r) protein (p180) is a substrate to the insulin receptor in vitro [Goren et al. (1990) Cellular Signalling 2, 537-555]. The question as to whether p180 is a substrate in vivo was addressed. Half ml 0.9% NaCl or 500 micrograms insulin was injected into rat livers. Purified plasma membrane glycoproteins from the livers were assayed for in vitro phosphorylation reaction products and endogenous tyrosine-phosphorylated proteins. Membranes from insulin-injected rat livers contained phosphorylated p180 and phosphorylated insulin receptor beta-subunit, whereas saline-injected rat liver membranes contained neither. These data suggested that p180 is an in vivo substrate to the insulin receptor. In vitro p180 is tyrosine-phosphorylated in the absence of insulin. p180, therefore, may be the epidermal growth factor (EGF) receptor or another tyrosine kinase that could be part of a phosphorylation cascade initiated by insulin. Two different experiments suggested that p180 is not the EGF receptor: (i) two-dimensional gel electrophoresis (first dimension--non-equilibrium pH-gradient gel electrophoresis) indicated that p180 is a more basic glycoprotein than EGF receptor; and (ii) based on reverse-phase high pressure liquid chromatography, the tryptic-phosphopeptides of carboxymethyl-Sepharose-purified phosphorylated-p180 were different from those of A431 cell phosphorylated-EGF receptor. Similarly, two different experiments demonstrated that p180 is not a tyrosine kinase: (i) gel-permeation chromatography separated the insulin receptor from p180 and only insulin receptor was autophosphorylated in vitro; and (ii) membrane proteins not bound to immobilized ATP contained p180. Thus, p180 can associate with the insulin receptor and be phosphorylated in vitro and in vivo; however, p180 does not function in an insulin receptor-mediated phosphorylation cascade.
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PMID:Plasma membrane p180, which insulin receptor phosphorylates in vivo, is not a tyrosine kinase. 834 20

The predicted human erbB-3 gene product is closely related to epidermal growth factor receptor (EGFR) and erbB-2, which have been implicated as oncogenes in model systems and human neoplasia. We expressed the erbB-3 coding sequence in NIH 3T3 fibroblasts and identified its product as a 180-kDa glycoprotein, gp180erbB-3. Tunicamycin and pulse-chase experiments revealed that the mature protein was processed by N-linked glycosylation of a 145-kDa erbB-3 core polypeptide. The intrinsic catalytic function of gp180erbB-3 was shown by its ability to autophosphorylate in vitro. Ligand-dependent signaling of its cytoplasmic domain was established employing transfectants that express a chimeric EGFR/erbB-3 protein, gp180EGFR/erbB-3. EGF induced tyrosine phosphorylation of the chimera and promoted soft agar colony formation of such transfectants. These findings combined with the detection of constitutive tyrosine phosphorylation of gp180erbB-3 in 4 of 12 human mammary tumor cell lines implicate the activated erbB-3 product in the pathogenesis of some human malignancies.
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PMID:Demonstration of ligand-dependent signaling by the erbB-3 tyrosine kinase and its constitutive activation in human breast tumor cells. 846 5

Using a panel of 20 non-small cell lung cancer (NSCLC) cell lines established from previously untreated patients, we investigated the relationships between intrinsic chemoresistance (to four agents used commonly in the therapy of NSCLC) and HER-2/neu gene expression (which encodes glycoprotein p185neu), p53 gene mutations, and cell proliferation characteristics. Our results demonstrated that high p185neu expression was correlated with chemoresistance, low S-phase fractions, and long doubling times. By contrast, cell lines expressing relatively low levels of p185neu were relatively chemosensitive and had higher S-phase fractions and shorter doubling times. Although mutation of the p53 gene was a common event in this panel of cell lines (present in 18 of 20 lines), there was no relationship between mutations at any specific codon and chemoresistance or cell proliferation characteristics. Multivariate analysis revealed that the level of p185neu was the only independent predictor for chemoresistance to doxorubicin, etoposide, and probably cisplatin. Although intrinsic chemoresistance almost certainly is a multifactorial process, overexpression of p185neu may be an important factor in the chemoresistance of NSCLC.
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PMID:Correlations between intrinsic chemoresistance and HER-2/neu gene expression, p53 gene mutations, and cell proliferation characteristics in non-small cell lung cancer cell lines. 854 64

The use of antibodies to target tumor antigens has had limited success, partially due to the large size of IgG molecules, difficulties in constructing smaller single chain Fv (scFv) antibody fragments, and immunogenicity of murine antibodies. These limitations can be overcome by selecting human scFv directly from non-immune or semi-synthetic phage antibody libraries; however, the affinities are typically too low for therapeutic application. For hapten antigens, higher-affinity scFv can be isolated from phage antibody libraries where the VH and VL genes of a binding scFv are replaced with repertoires of V genes (chain shuffling). The applicability of this approach to protein binding scFv is unknown. For this work, chain shuffling was used to increase the affinity of a non-immune human scFv, which binds the glycoprotein tumor antigen c-erbB-2 with an affinity of 1.6 x 10(-8) M. The affinity of the parental scFv was increased sixfold (Kd = 2.5 x 10(-9) M) by light-chain shuffling and fivefold (Kd = 3.1 x 10(-9) M) by heavy-chain shuffling, values comparable to those for antibodies against the same antigen produced by hybridomas. When selections were performed on antigen immobilized on polystyrene, spontaneously dimerizing scFv were isolated, the best of which had only a slightly lower Kd than wild type (Kd = 1.1 x 10(-8) M). These scFv dimerize on phage and are preferentially selected as a result of increased avidity. Compared to scFv which formed only monomer, dimerizing scFv had mutations located at the VH-VL interface, suggesting that VH-VL complementarity determines the extent of dimerization. Higher-affinity monomeric scFv were only obtained by selecting in solution using limiting concentrations of biotinylated antigen, followed by screening mutant scFv from bacterial periplasm by koff in a BIAcore. Using the proper selection and screening conditions, protein binding human scFv with affinities comparable to murine hybridomas can be produced without immunization.
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PMID:Isolation of high-affinity monomeric human anti-c-erbB-2 single chain Fv using affinity-driven selection. 856 73

For in vitro evolution of protein function, we previously proposed using parsimonious mutagenesis (PM), a technique where mutagenic oligodeoxynucleotides (oligo) are designed to minimize coding sequence redundancy and limit the number of amino acid (aa) residues which do not retain parental structural features. For this work, PM was used to increase the affinity of C6.5, a human single-chain Fv (scFv) that binds the glycoprotein tumor antigen, c-erbB-2. A phage antibody library was created where 19 aa located in three of the heavy (H) and light (L) chain antigen-binding loops (L1, L3 and H2) were simultaneously mutated. After four rounds of selection, 50% of scFv had a lower dissociation rate constant (koff) than the parental scFv. The Kd of these scFv ranged from twofold (Kd=7.0 x 10(-9) M) to sixfold (Kd=2.4 x 10(-9) M) lower than the parental scFv (Kd=1.6 x 10(-8) M). In higher affinity scFv, substitutions occurred at 10/19 of the positions, with 21/28 substitutions occurring at only four positions, two in H2, and one each in L1 and L3. Only the wild type (wt) aa was observed at 9/19 aa. Based on a model of C6.5, seven of the nine conserved aa have a structural role in the variable domain, either in maintaining the main chain conformation of the loop, or in packing on the H-chain variable domain. Two of the conserved aa are solvent exposed, suggesting they may play a critical role in recognition. Thus, PM identified three types of aa: structural aa, functional aa which modulate affinity, and functional aa, which are critical for recognition. Since the sequence space was not completely sampled, higher affinity scFv could be produced by subjecting functional aa which modulate affinity to a higher rate of mutation. Furthermore, PM could prove useful for modifying function in other proteins that belong to structurally related families.
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PMID:Identification of functional and structural amino-acid residues by parsimonious mutagenesis. 864 39

Previously, a panel of mouse monoclonal antibodies (mAbs) to several tumor-associated antigens was chemically crosslinked to an IgG1 anti-human transferrin receptor antibody, 454A12. We called this new class of bispecific antibodies (BmAbs) "antigen forks" and showed that these antigen forks inhibited but did not completely prevent tumor cell growth. We speculated that the conjugates acted by heterologously crosslinking two antigens in a manner that interfered with the functions of one or both. The most effective BmAbs all shared one specificity for the human transferrin receptor. A monoclonal antibody to this receptor has been shown by others to reduce tumor cell growth when used with the iron chelator deferoxamine. When we combined our antigen forks with deferoxamine, two of five BmAbs synergized with deferoxamine to arrest tumor cell count at or below input levels. The most effective BmAbs were 317G5/454A12 (3/4) and 520C9/454A12 (5/4). mAb 317G5 recognizes a 42-kDa tumor-associated glycoprotein, and mAb 520C9 recognizes the c-erbB-2 protooncogene product. BmAb 3/4 was most effective against colorectal cancer cell line HT-29, and BmAb 5/4 was most effective against breast cancer cell line SK-BR-3. When deferoxamine and BmAb were replaced by fresh medium after a 6- or 7-day treatment period, no regrowth of tumor cells was observed during the next 4 days, although regrowth was seen if either deferoxamine or BmAb was used alone. Our results show that BmAbs with specificities for transferrin receptor and certain tumor-associated antigens effectively inhibit tumor growth in vitro. When used in combination with deferoxamine, such BmAbs may have therapeutic potential for the treatment of cancer.
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PMID:In vitro tumor growth inhibition by bispecific antibodies to human transferrin receptor and tumor-associated antigens is augmented by the iron chelator deferoxamine. 876 64


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