In vivo evaluation of epidermal growth factor (EGF) receptor density on human tumor xenografts using radiolabeled EGF and anti-(EGF receptor) mAb 425

 In order to study the potential of non-invasive scintigraphic evaluation of the epidermal growth factor (EGF) receptor status in vivo, the biokinetics and tumor binding of ¹²⁵I-EGF and anti-(EGF receptor) mAb 425 were investigated in nude mice bearing human tumor xenografts with different EGF-receptor densities as determined by a radioreceptor assay. The results demonstrated a tumor uptake for both substances depending on the receptor level. The EGF receptor status, however, was reflected slightly better by the binding of EGF to tumor tissue compared to the mAb. The rapid blood clearance of EGF with a plasma half-life of less than 1 min led to a tumor-to-blood ratio of approximately 3 within 6 h after injection in tumors with a high receptor expression. A similar ratio for the mAb was not obtained before day 6 after injection. The absolute concentration of EGF, however, was low compared to the mAb. Therefore, it can be concluded that the EGF receptor status as a target for (radio)immunotherapy can be evaluated in vivo with EGF labeled with a short-life positron-emitting radionuclide or with monoclonal antibodies to the EGF receptor or their fragments. Received: 14 September 1995 / Accepted: 6 December 1995     

Therapeutic potential of chimeric anti-(ganglioside GD3) antibody KM871: antitumor activity in xenograft model of melanoma and effector function analysis

KM871 is a chimeric antibody recognizing ganglioside GD3, which is one of the major gangliosides expressed on the cell surface of human tumors of neuroectodermal origin. This study demonstrates the antitumor activity of KM871 against human melanoma xenografts in nude mice, and analyzes the effector function operating in mice. In a well-established tumor model, KM871 showed antitumor activity against H-15 and SK-MEL-28 human melanoma but not against H-187 and G361 human melanoma when administered intravenously 5 days/week for 2 weeks. The G361 tumor became sensitive when KM871 was first administered on the day of tumor inoculation. In this assay, it was observed that almost all the mice were tumor-free, but a few mice developed tumors. Therefore, we examined the amount and expression pattern of GD3 antigen on G361 tumors escaping from KM871 treatment, but no change was observed. Next we examined the optimal administration schedule for KM871 in mice, using H-15 melanoma. KM871 showed antitumor activity when administered intravenously either 5 days/week for 2 weeks or three biweekly doses. However, the effect of the former schedule was stronger than three biweekly doses. To compare the effector function in humans and mice, we studied the complement-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity and antibody-dependent macrophage-mediated cytotoxicity of KM871 using complement or effector cells prepared from humans and mice. It was found that the antibody-dependent cell-mediated cytotoxicity exerted by polymorphonuclear cells and antibody-dependent macrophage-mediated cytotoxicity were the only antitumor mechanism of KM871 in mice. However their action was very weak compared with that in humans, and complement-mediated cytotoxicity, which was strong in humans, was not observed in mice. Therefore, the antitumor activity of KM871 against human melanomas evaluated by the nude mouse model might be underestimated. These results indicate that KM871 shows good antitumor activity against GD3-positive human melanoma and the antitumor activity expected in humans might be superior to that of the nude mouse model. Received: 10 July 1999 / Accepted: 21 January 2000     

A mouse/human chimeric anti-(ganglioside GD3) antibody with enhanced antitumor activities

Ganglioside GD3, which is one of the major gangliosides expressed on the cell surface of human tumors of neuroectodermal origin has been focused on as a target molecule for passive immunotherapy. We have cloned the cDNA encoding the immunoglobulin light and heavy chains of an anti-GD3 monoclonal antibody KM641 (murine IgG3, ), and constructed the chimeric genes by linking the cDNA fragments of the murine light and heavy variable regions to cDNA fragments of the human and 1 constant regions, respectively. The transfer of these cDNA constructs into SP2/0 mouse myeloma cells resulted in the production of the chimeric antibody, designated KM871, that retained specific binding activity to GD3. Indirect immunofluorescence revealed the same staining pattern for chimeric KM871 and the mouse counterpart KM641 on GD3-expressing melanoma cells. When human serum and human peripheral blood mononuclear cells were used as effectors in complement-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity respectively, the chimeric KM871 was more effective in killing GD3-expressing tumor cells than was the mouse counterpart KM641. Intravenous injection of chimeric KM871 markedly suppressed tumor growth in nude mice. The chimeric KM871, having enhanced antitumor activities and less immunogenicity than the mouse counterpart, would be a useful agent for passive immunotherapy of human cancer.     

Activation mechanism of solubilized epidermal growth factor receptor tyrosine kinase.

Dimerization of epidermal growth factor receptor (EGFR) leads to the activation of its tyrosine kinase. To elucidate whether dimerization is responsible for activation of the intracellular tyrosine kinase domain or just plays a role in the stabilization of the active form, the activated status of wild-type EGFR moiety in the heterodimer with kinase activity-deficient mutant receptors was investigated. The kinase activity of the wild-type EGFR was partially activated by EGF in the heterodimer with intracellular domain deletion (sEGFR) or ATP binding-deficient mutant (K721A) EGFRs, while the wild-type EGFR in the heterodimer of wild-type and phosphate transfer activity-deficient mutant receptor D813N could be fully activated. After treatment with EGF, the ATP binding affinity and the V(max) of the wild-type EGFR increased. In the presence of sEGFR, a similar increase in the affinity for ATP was observed, but V(max) did not change. A two-step activation mechanism for EGFR was proposed: upon binding of EGF, the affinity for ATP increased and then, as a result of interaction between the neighboring tyrosine kinase domain, V(max) increased.     

Increased epidermal growth factor receptor in multidrug-resistant human neuroblastoma cells

Multidrug-resistant human neuroblastoma cell lines obtained by selection with vincristine or actinomycin D from two independent clonal lines, SH-SY5Y and MC-IXC, have 3- to 30-fold more cell surface epidermal growth factor (EGF) receptors than the drug-sensitive parental cells as indicated by EGF binding assays and immunoprecipitation, affinity-labeling, and phosphorylation studies. Reversion to drug sensitivity in one line was accompanied by a return to the parental level of EGF receptor. SH-EP cells, a clone derived from the same neuroblastoma cell line as SH-SY5Y but which displays melanocyte rather than neuronal lineage markers, also express significantly more EGF receptor than SH-SY5Y cells. By nucleic acid hybridization analysis with a molecularly cloned probe, increased receptor level in multidrug-resistant cells was shown to be the result of higher levels of EGF receptor mRNA in drug-resistant than in drug-sensitive cells. The increased steady state amount of specific RNA did not result from amplification of receptor-encoding genes. A small difference was observed in the electrophoretic mobility under denaturing conditions of EGF receptor immunoprecipitated from drug-resistant and drug-sensitive cells. Quantitative and qualitative modulation of the EGF receptor might reflect alterations in the transformation and/or differentiation phenotype of the resistant cells or might result from unknown selective pressures associated with the development of multidrug resistance.     

Localization of the epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) in the bovine testis

In the last few decades, several growth factors were identified in the testis of various mammalian species. Growth factors are shown to promote cell proliferation, regulate tissue differentiation, and modulate organogenesis. In the present investigation we have studied the localization of EGF and EGFR in the adult bovine testis by means of immunohistochemical method. Our results demonstrated that EGF and EGFR were localized solely to the bovine testicular germ cells (spermatogonia, spermatocytes, and round spermatids). In contrast, the somatic testicular cells (i.e., Sertoli, Leydig, and myofibroblast cells) exhibited no staining affinity. EGF and EGFR were additionally detected in the epithelial lining of straight tubules and rete testis. Interestingly, the distribution of EGF and EGFR in the germ cells was mainly dependent upon the cycle of the seminiferous epithelium since their localization appeared to be preponderant during the spermatogonia proliferation and during the meiotic and spermiogenic processes. In conclusion, such findings may suggest that EGF and EGFR are important paracrine and/or autocrine regulators of spermatogenesis in bovine.     

Involvement of the signal transduction pathway mediated by epidermal growth factor receptor in the differentiation of chicken glandular stomach

During the development of the chicken proventriculus (glandular stomach), the initially undifferentiated epithelium differentiates into two distinct cell populations: the glandular epithelium, cells of which secrete embryonic chicken pepsinogen (ECPg), and luminal epithelial cells, which express the chicken spasmolytic polypeptide gene (cSP). Based on knowledge of the adult mouse stomach, the ligands of epidermal growth factor (EGF) receptor (EGFR) were expected to affect differentiation of the proventricular epithelium. When EGF was added to the medium in which proventriculi were cultured in vitro, gland formation was suppressed in a dose-dependent manner and the amount of ECPg mRNA decreased, whereas morphological differentiation of luminal epithelium was stimulated. Simultaneous treatment of the proventriculus with EGF and tyrphostin 47 resulted in the attenuation of the effect of EGF, suggesting that EGF, or other ligands of EGFR, may actually be involved in the normal course of development of the proventricular epithelium.     

Modulation of the epidermal growth factor receptor by brain-derived growth factor in Swiss mouse 3T3 cells

Incubation of Swiss mouse 3T3 cells at 37 degrees C with bovine brain-derived growth factor (BDGF) decrease the cell surface 125I-EGF binding activity of these cells by 70-80%. This down-modulation of the EGF receptor by BDGF was time, temperature, and dose dependent. Scatchard plot analysis indicated that BDGF binding led to a selective decrease in the number of high-affinity EGF receptors. The BDGF-induced down-modulation of the EGF receptor was completely blocked by protamine, a potent inhibitor of receptor binding and mitogenic activities of BDGF. BDGF down-modulated the EGF receptor in phorbol myristic acetate (PMA)-pretreated cells, as well as in control cells. Furthermore, PMA-pretreated cells responded mitogenically to BDGF, whereas PMA itself failed to stimulate the mitogenic response of PMA-pretreated cells. This BDGF-induced down-modulation of the EGF receptor in PMA-desensitized cells suggests that BDGF down-regulates the EGF receptor by a mechanism distinct from that of PMA. Incubation of cells with compounds which are known to inhibit pinocytosis blocked the down-modulation induced either by BDGF or by platelet-derived growth factor (PDGF) but had no effect on the PMA-induced down-modulation. Incubation of cells with inhibitors of receptor recycling enhanced the BDGF-induced down-modulation of the EGF receptor. These results suggest that BDGF and PDGF induce down-modulation of the EGF receptor by increasing the internalization of cell surface high-affinity receptors and that the internalization process may not be required for down-modulation induced by PMA.     

Modulation of drug-induced cytotoxicity by a bispecific monoclonal antibody that recognizes the epidermal growth factor receptor and doxorubicin

A hybrid hybridoma secreting a bispecific hybrid mAb (bsmAb), which recognizes both the epidermal growth factor receptor (EGF-R) and the drug doxorubicin, was produced by somatic hybridization of two hybridomas. The bsmAb obtained was able to retarget doxorubicin cytotoxicity in vitro specifically on EGF-R-positive cells exerting at the same time an antidotal effect on cells that did not overexpress the EGF-R. Distribution studies in mice indicate that the bsmAb selectively delivers the drug to tumour cells and modifies doxorubicin biodistribution with a statistically significant decrease of drug concentration in the intestine, which is the main target of early anthracycline toxicity. In keeping with this finding is the remarkable antidotal activity exerted by bsmAb in mice treated with doxorubiein, which is proved by retardation in loss of body weight and mortality. The effectiveness on tumour growth of the mAb followed by the administration of doxorubicin appears to be equal to that of the drug alone; however, the bsmAb exerts a remarkable antidotal activity.     

嵌合抗原受体T细胞治疗血液肿瘤的研究进展

嵌合抗原受体T细胞免疫疗法(chimeric antigen receptor T-cell immunotherapy,CAR-T)是近年来迅速发展的肿瘤过继免疫治疗方法,其胞外段抗体以非主要组织相容性复合物(major histocompatibility complex,MHC)方式与相应的肿瘤相关抗原结合识别后,使T细胞活化而发挥抗肿瘤效应。CAR-T在血液疾病治疗中取得较好的效果,现就CAR的结构、CAR-T治疗的靶点、出现的不良反应及采取的相应策略等作一概述。     

Modulation of mouse preimplantation development by epidermal growth factor receptor antibodies,antisense RNA,and deoxyoligonucleotides

Two-cell mouse preimplantation embryos were cultured for 48 h in four different reagents to modulate epidermal growth factor (EGF) receptor function. These were rabbit polyclonal and mouse monoclonal antibodies to EGF receptor, EGF receptor antisense RNA, and EGF receptor antisense deoxyoligonucleotides. Embryos were scored for two endpoints: onset of cavitation as a measure of trophectoderm differentiation and mean embryo cell number as a measure of cell proliferation. The consistent observations were that cavitation was significantly accelerated by antibodies and delayed by antisense RNA and antisense deoxyoligonucleotides. None of these reagents exerted a significant effect on mean embryo cell number, with one exception the polyclonal antibody. Our interpretation of these observations is that the antibody binding facilitated cavitation by mimicking natural ligand-receptor binding and inducing the signal transduction cascade that is typical for the EGF receptor. In the case of antisense RNA or deoxyoligonucleotide, we propose that they delayed onset of cavitation by interfering with EGF receptor production. We hypothesize that during this period of development, EGF receptor is concerned predominantly with the regulation of differentiation more than with cell proliferation. © 1993Wiley-Liss, Inc.     

Presence and localization of epidermal growth factor (EGF)- and EGF-receptor-like immunoreactivity in Tetrahymena

The presence of EGF- and EGF-receptor-like immunoreactivity in Tetrahymena was studied with the help of FITC-labelled monoclonal antibodies, using flow cytometry and confocal microscopy. Tetrahymena has endogeneous EGF and treatment with it significantly increases the hormone content of the cells. The hormone is diffusely localized, particularly in the cytopharynx, where it forms a structure larger than 10 microm. EGF-receptors are also demonstrable, particularly in the cortical region in connection with cilia, and EGF treatment significantly enhances cortical fluorescence. The relationships between these observations and literary data on the effects of EGF in Tetrahymena are discussed.     

Effect of epidermal growth factor on preimplantation development and its receptor expression in porcine embryos.

The present study aimed to determine the influence of exogenous epidermal growth factor (EGF) on in vitro preimplantation porcine embryo development and its mRNA expression for EGF receptor (EGFR). Oocytes were aspirated from abattoir ovaries, selected and cultured in defined, protein-free media for 44 hr before in vitro fertilization (IVF). Thirty-six hours after IVF, two-cell stage embryos were selected and treated or cultured until embryo treatment. In experiment 1, compact morulae were selected on day 4 after IVF and randomly allocated into 5 groups: NCSU 23 with PVA as group 1; NCSU 23 with PVA and 0.1 ng/ml, 1.0 ng/ml, 10.0 ng/ml EGF as group 2, 3, 4, respectively; NSCU 23 with 0.4% BSA as group 5. In experiment 2, treatment groups were the same as in experiment 1 except that 0.1% crystallized BSA was added to both washing media and all treatment groups instead of PVA. In experiments 3 and 4, two-cell stage embryos were treated and cultured in the same experimental design as experiments 1 and 2, respectively. RT-PCR was used to detect the mRNA expression of EGF receptor in compact morulae and blastocysts. The PCR products were subjected to direct DNA sequencing. There was no significant improvement in the development rate of embryos from compact morulae to blastocysts in the presence of various EGF concentrations (0.1, 1.0, 10.0 ng/ml) versus without EGF addition. They were all significantly lower than those embryos cultured in the continuous presence of 0.4% BSA. However, when a reduced concentration (0.1%) of crystallized BSA was added to all the treatment groups, a significantly lower rate of embryo development was observed in control media (NCSU23 with 0.1% crystallized BSA) compared with those developed in culture media with 0.4% BSA. With the addition of EGF at 10 ng/ml (with 0.1% BSA), embryo development rates were significantly improved over the control group (P < 0.05) and were as good as those rates in 0.4% BSA culture group. When embryos were selected and treated from the 2-cell stage, they did not develop to blastocyst stages after five more days' culture without any protein (BSA) or growth factor addition. When 0.1% BSA was included in the media, blastocyst formation rates were significantly improved by EGF addition at the concentration of both 1.0 or 10 ng/ml (P < 0.05) as compared to 0.0 or 0.1 ng/ml. EGFR mRNA was detected in both compact morulae and blastocyst stages of porcine embryos and confirmed by direct DNA sequencing. Our results indicate that IVM-IVF porcine embryo developmental rates could be improved by the addition of EGF in the culture media with the presence of a reduced amount of defined BSA (>97% albumin). However, EGF alone was not able to elicit any stimulatory effects on embryo development in the absence of protein supplementation. Further studies are needed to investigate the potential synergistic factors in embryo culture media to eventually define the porcine embryo culture media.     

Effect of cycloheximide on epidermal growth factor receptor trafficking and signaling

Cycloheximide is the most common protein synthesis inhibitor, and is believed to specifically inhibit the cytoplasmic protein synthesis. Here we demonstrate that cycloheximide induces internalization and redistribution of EGF receptor to early endosomes in HeLa cells independent of receptor tyrosine phosphorylation, but dependent on p38 MAPK activity. Degradation of EGF receptor or its downstream effectors was not observed. EGF-induced activation of ERK1/2 was inhibited upon pre-treatment with cycloheximide, but did not activate JNK. The observed effects of treatment with cycloheximide alone are significant and therefore results involving the use of cycloheximide for inhibition of protein synthesis must be interpreted with caution.

Structured summary of protein interactions

EEA1 and EGFRcolocalize by fluorescence microscopy (View interaction).     

Novel monoclonal antibodies recognizing the active conformation of epidermal growth factor receptor

The precise regulation of epidermal growth factor receptor (EGFR) is crucial for its function in cellular growth control. Although many antibodies against EGFR have been developed and used to analyze its regulation and function, it is not yet easy to analyze activated EGFR specifically. Activated EGFR has been mainly detected by its phosphorylation state using anti-phospho-EGFR and anti-phosphotyrosine antibodies. In the present study, we have established novel monoclonal antibodies which recognize the activated EGFR independently of its phosphorylation. Our antibodies detected active state of EGFR in immunoprecipitation and immunofluorescence, by recognizing the epitopes which are exposed through the conformational change induced by ligand-binding. Furthermore, we found that our antibodies preferentially detected the conformation of constitutively active EGFR mutants found in lung cancer cell lines. These results indicate that our antibodies may become novel research and diagnostic tools for detecting and analyzing the conformation of active EGFR in various cells and tissues.     

Immunoreactivities for epidermal growth factor (EGF) and for EGF receptors in rats with gastric ulcers

Summary The present study was aimed at assessing whether epidermal growth factor (EGF) and its receptors are present in the gastric mucosa during the healing of gastric ulcers. Immunohistochemical, immunochemical and functional studies were performed in rats after induction of ulcers in the oxyntic mucosa. Controls, which included non-operated and sham-operated animals, displayed only rare cells in the bottom of the oxyntic glands showing EGF-like immunoreactivity. Within one day after ulcer induction, a markedly increased number of chief cells in undamaged mucosa showed intense staining. Concomitantly, there was an increased immunoreactivity for EGF receptors in the mucous neck cells. Maximal immunostaining for both compounds was observed at 3 days after ulcer induction; augmented staining was still demonstrable after 3 weeks. RIA revealed significantly increased EGF concentration in the oxyntic mucosa three days after ulcer induction, and at this stage stimulated gastric acid secretion, measured in a parallel group of chronic fistula rats, indicated significant inhibition. The transient increases in EGF-like and EGF receptor immunoreactivities may stimulate gland cell proliferation. The local release of EGF-like substances may also serve to reduce gastric acidity and thereby promote ulcer healing.