异种表皮生长因子受体胞外段的表达及其抗肿瘤效果研究

目的:构建异种EGFR蛋白疫苗并研究其抗肿瘤效果.方法:用PCR方法获得鸡EGFR胞外片段, 与pGEX- 4T-2载体连接, 并在E.coli BL21(DE3)中表达鸡的EGFR胞外片段与GST的融合蛋白, 融合蛋白经Ni2 亲和层析柱纯化、复性后, 免疫小鼠, 免疫3次后, 接种小鼠Lewis细胞, 观测肿瘤生长情况, ELISA检测小鼠血清中抗EGFR蛋白的抗体效价.结果:成功构建了EGFR胞外段基因的原核表达载体; SDS-PAGE显示成功表达了目的融合蛋白; 融合蛋白免疫小鼠后, ELISA法检测到特异性抗EGFR抗体; 实验组与对照组肿瘤体积的比较结果显示, 融合蛋白疫苗能够在一定程度上抑制肿瘤的生长.结论:异种EGFR蛋白能够克服免疫耐受问题, 诱导机体产生抗体, 有一定的抗肿瘤效果, 为后续的肿瘤疫苗研究打下了基础.     

Expression of epidermal growth factors and epidermal growth factor receptor in normal cycling human ovaries

Immunolocalization of transforming growth factor- (TGF), epidermalgrowth factor (EGF), cripto-1, amphireg-ulin and epidermal growthfactor receptor (EGFR ) was studied in 51 premenopausal humanovaries at various phases of the menstrual cycle. Localizationof mRNA for TGF and EGF was also studied by in-situ hybridization.Immunoreactive TGF was observed predominantly in theca cellsin 12 of 33 antral follicles in the follicular phase (6/14 dominantfollicles, and 6/19 non-dominant) but not in any of the 18 folliclesin the luteal phase or in primordial and pre-antral follicles.TGF immunoreactivity was present predominantly in the luteinizedgranulosa cells in 13 of 15 corpora lutea in the luteal phase,which are considered to be active in steroidogenesis, but notin any of the regressed corpora lutea. Accumulation of TGF mRNAhybridization signal was observed only in the theca cells inthe follicles and luteinized theca cells in the ovaries thatwere immunohistochemically positive for TGF. EGFR immunoreactivitywas detected in 24 of 33 antral follicles in the follicularphase and in two of 18 follicles in the luteal phase but notin any of the corpora lutea. Immunoreactive EGF, cripto-1 andamphiregulin or EGF mRNA was not detected in any follicles,corpora lutea, or the stroma cells examined. These results indicatethat, of the epidermal growth factors examined in this study,TGF is locally synthesized in normal cycling human ovaries andTGF may be synthesized in theca cells and act on the granulosacells in a paracrine fashion through the EGFR in ovarian follicles. EGF family/human/immunohistochemistry/in-situ hybridization/ovary     

抗重组人表皮生长因子单克隆抗体的制备及生物学特性鉴定

目的　制备出高效价的抗重组人表皮生长因子 (EGF)单克隆抗体。方法　以重组人表皮生长因子作为抗原 ,免疫Balb/c小鼠 ,以未免疫的Balb/c小鼠脾细胞为饲养细胞 ,运用细胞杂交瘤技术制备 ,间接ELISA法筛选产生针对人表皮生长因子的单克隆抗体细胞株 ;以体内诱生法产生腹水 ,并采用ProteinA Sepharose柱对其纯化 ,快速定性试纸鉴定McAb的Ig亚类 ,采用间接ELISA法相加实验鉴定抗原识别表位。结果　获得 3株产生针对人表皮生长因子的单克隆抗体细胞株EGF B2 、EGF C7、EGF A8,Ig亚分别为IgG1κ型 ,IgG1λ型 ,IgG3 κ型 ,亲和力常数分别为 2 .76× 10 10 、3.2× 10 9、1.4 5× 10 9。结论　成功制备 3株稳定分泌抗rhEGF的杂交瘤细胞株 ,产生的McAb特异性好 ,亲和力高 ,为探讨EGF的作用机制及临床应用奠定了基础 ,为肿瘤的诊断与治疗提供具有实用价值的研究工具 ,此外 ,为EGF的纯化提供实验材料     

Expression of epidermal growth factor and its receptor in rabbits with ischaemic acute renal failure

Urinary immunoreactive epidermal growth factor (EGF) levels decrease, and renal immunoreactive EGF levels increase in rats with ischaemic acute renal failure (ARF). We investigated the immunohistochemical localization of EGF and EGF receptor in rabbits with ischaemic ARF to clarify the significance of renal EGF. Male New Zealand White rabbits underwent right nephrectomy prior to a 60 min renal artery clamp. At 3, 6, 24, 48, 72 and 96 h after ischaemia, serum urea nitrogen and serum creatinine were determined. Guinea pig anti-rabbit EGF antibody and monoclonal anti-EGF receptor antibody were used for the primary incubation. EGF was immunolocalized to the ascending limb of Henle and the distal convoluted tubule in the normal right kidneys. However, in the post ischaemic left kidneys at 6, 24, 48 and 72 h, immunoreactivity of EGF was associated with proximal tubules. In the normal kidneys, antibody to EGF receptor reacted with distal tubules and collecting ducts. In the ischaemic kidneys, EGF receptor was localized in the basolateral membrane in the proximal tubules. The expression of EGF and EGF receptor in renal tubules may play an important role in repair following ischaemic renal damage.     

Expression of epidermal growth factor receptor in breast carcinoma.

A series of 90 patients with primary operable breast cancer and with up to 36 months of follow up were investigated for expression of epidermal growth factor receptor (EGFR) in their tumours by immunocytochemical staining with the monoclonal antibody EGFR 1. Tumour samples were snap frozen in liquid nitrogen immediately after resection and subsequently stained using a standard indirect immunocytochemical method. Tumour staining was assessed by two observers and scored on a four point scale (0-3). Thirteen (14%) tumours showed positive immunoreactivity. A strong correlation between distinct EGFR expression and short disease free interval was observed. Significant correlations were also shown with oestrogen and progesterone receptor expression and tumour nuclear size. No significant association was found with tumour size, lymph node stage, and histological grade. The association with disease free interval remained significant in multivariate analysis.     

Expression of epidermal growth factor receptor and transferrin receptor by human trophoblast populations

Epidermal growth factor (EGF) has several roles, including stimulation of cell division and differentiation. EGF receptor (EGFR) has been localized to villous syncytiotrophoblast, but expression by other human trophoblast populations has not been reported. EGFR expression was examined in normal and pathological placental tissues using a streptavidin-biotin-peroxidase technique; results were compared with expression of transferrin receptor (Tf-R) in similar tissues. EGFR was detected on villous syncytiotrophoblast in early and term pregnancy with labelling of the apical membrane, focal cytoplasmic reactivity, and patchy labelling of the trophoblast basement membrane. In contrast with other reports, EGFR was also consistently localized to villous cytotrophoblast, chorion laeve, and extravillous trophoblast populations in maternal uterine tissues. Maternal decidua showed diffuse labelling of stromal cells, particularly in the superficial zones. The reaction pattern in ectopic tubal pregnancy was similar to that in early intrauterine pregnancy. In molar pregnancy, EGFR was detected on villous syncytiotrophoblast and cytotrophoblast. In contrast, in normal, ectopic, and molar pregnancies labelling for Tf-R was confined to syncytiotrophoblast and to the proximal portions of the cytotrophoblast columns. Expression of EGFR by all trophoblast cells may represent a mechanism of placental growth and proliferation control. EGFR may also be involved with establishment of differentiated trophoblast functions including hormone secretion.     

Reactivity of epidermal growth factor receptor monoclonal antibodies with human uterine tissues

Two monoclonal antibodies (29.1 and 528), which were raised against the epidermal growth factor receptor, were used to evaluate expression of epidermal growth factor receptor in frozen uterine tissue with immunohistochemical techniques. Monoclonal antibody 29.1 stained only vascular endothelium and glandular epithelium in patients who were blood type A. This staining pattern is consistent with the previously reported blood type A specificity of this antibody. Monoclonal antibody 528, which recognizes a peptide determinant is thought to be specific for the epidermal growth factor receptor, stained both endometrial glands and endometrial stromal cells heavily. Faint staining was also seen in myometrium in most cases. This marked difference in expression of epidermal growth factor receptor between endometrium and myometrium contrasts with results of prior biochemical studies in which tissue homogenates were used. No variation in intensity of staining was seen between proliferative and secretory endometrium with the use of either antibody.     

Heparin-binding epidermal growth factor cleavage mediates zinc-induced epidermal growth factor receptor phosphorylation

We have previously shown that exposure to zinc ions can activate epidermal growth factor (EGF) receptor (EGFR) signaling in murine fibroblasts and A431 cells through a mechanism involving Src kinase. While studying the effects of zinc ions in normal human bronchial epithelial cell, we uncovered evidence for an additional mechanism of Zn(2+)-induced EGFR activation. Exposure to Zn(2+) induced phosphorylation of EGFR at tyrosine 1068, a major autophosphorylation site, in a dose- and time-dependent fashion. This effect of Zn(2+) on EGFR was significantly blocked with an antibody against the ligand-binding domain of the receptor. Neutralizing antibodies against EGFR ligands revealed the involvement of heparin-binding EGF (HB-EGF) in Zn(2+)-induced EGFR phosphorylation. This observation was further supported by immunoblots showing elevated levels of HB-EGF released by Zn(2+)-exposed cells. Zymography showed the existence of matrix metalloproteinase-3 in Zn(2+)-challenged cells. Incubation with a specific matrix metalloproteinase-3 inhibitor suppressed Zn(2+)-induced EGFR phosphorylation as well as HB-EGF release. Therefore, these data support an autocrine or paracrine mechanism whereby Zn(2+) induces EGFR phosphorylation through the extracellular release of EGFR ligands, which may be mediated by metalloproteinases.     

Endocrinology and paracrinology: Expression of epidermal growth factors and epidermal growth factor receptor in normal cycling human ovaries

Immunolocalization of transforming growth factor- (TGF), epidermalgrowth factor (EGF), cripto-1, amphiregulin and epidermal growthfactor receptor (EGFR ) was studied in 51 premenopausal humanovaries at various phases of the menstrual cycle. Localizationof mRNA for TGF and EGF was also studied by in-situ hybridization.Immunoreactive TGF was observed predominantly in theca cellsin 12 of 33 antral follicles in the follicular phase (6/14 dominantfollicles, and 6/19 non-dominant) but not in any of the 18 folliclesin the luteal phase or in primordial and pre-antral follicles.TGF immunoreactivity was present predominantly in the luteinizedgranulosa cells in 13 of 15 corpora lutea in the luteal phase,which are considered to be active in steroidogenesis, but notin any of the regressed corpora lutea. Accumulation of TGF mRNAhybridization signal was observed only in the theca cells inthe follicles and luteinized theca cells in the ovaries thatwere immunohistochemically positive for TGF. EGFR immunoreactivitywas detected in 24 of 33 antral follicles in the follicularphase and in two of 18 follicles in the luteal phase but notin any of the corpora lutea. Immunoreactive EGF, cripto-1 andamphiregulin or EGF mRNA was not detected in any follicles,corpora lutea, or the stroma cells examined. These results indicatethat, of the epidermal growth factors examined in this study,TGF is locally synthesized in normal cycling human ovaries andTGF may be synthesized in theca cells and act on the granulosacells in a paracrine fashion through the EGFR in ovarian follicles.     

Monoclonal antibody to human epidermal growth factor

A monoclonal antibody directed to a species-specific determinant of human epidermal growth factor (h-EGF) was obtained by fusing murine myeloma cells with BALB/c mouse splenocytes sensitized to h-EGF. This antibody, referred to as 863.D4, did not react with either rat or mouse epidermal growth factor or with 11 other polypeptide hormones tested as shown by solid-phase radioimmunoassay (SPRIA), and immunoprecipitation followed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Scatchard analysis of the antibody binding to purified h-EGF revealed an apparent equilibrium dissociation constant of 1 X 10(-8) M. The antibody blocked both the binding of h-EGF and h-EGF stimulation of 3H-thymidine incorporation into DNA by greater than 90% in confluent cultures of human foreskin fibroblasts.     

No association between epidermal growth factor and epidermal growth factor receptor polymorphisms and nasopharyngeal carcinoma

Numerous candidate genes have been proposed as susceptibility factors for the development of nasopharyngeal carcinoma (NPC). Epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) interaction plays a pivotal role in cell proliferation, differentiation, and tumourigenesis of epithelial tissues. To our knowledge, however, no study has examined the relationship between the EGF/EGFR and NPC. The aim of this study is to investigate the potential association between single-nucleotide polymorphisms of EGF +61 G/A and EGFR +2073 A/T and NPC. A total of 173 patients with NPC and 206 age- and sex-matched controls were the participants. Genotypes were determined using a polymerase chain reaction-restriction fragment length polymorphism strategy and DNA sequencing. There were no significant differences in the genotype and allele frequencies of EGF +61 G/A and EGFR +2073 A/T polymorphisms between the group of patients with NPC and the control group in a Chinese population (for EGF +61 G/A: OR=1.29, 95% CI: 0.95-1.74; for EGFR +2073 A/T: OR=0.91, 95% CI: 0.67-1.23). Further studies are still needed to explore the complicated interaction between environmental factors and EGF +61 G/A and EGFR +2073 A/T polymorphisms in the risk of NPC, particularly in ethnically different populations.     

Expression of epidermal growth factor receptor in human meningiomas and meningeal tissue.

The aim of this study was to examine meningeal tissue under normal, reactive, and neoplastic conditions for expression of epidermal growth factor receptor (EGFR) using an improved histochemical method, namely biotinylated epidermal growth factor. EGFR was found in all the examined meningiomas (12 benign and three anaplastic) and in neonatal rat meninges, whereas normal and injured adult human and rat meninges did not exhibit detectable EGFR. These observations indicate that EGFR is involved in the development of meningeal tissue. Further, EGFR is abnormally expressed in meningeal tumours, indicating a role of EGFR in the neoplastic process of these tumours. The regular expression of EGFR in human meningiomas suggests EGFR as a tumour marker for this tumour type.     

Analysis of epidermal growth factor receptor and activated epidermal growth factor receptor expression in pituitary adenomas and carcinomas.

Epidermal growth factor receptor plays an important role in the pathogenesis of many malignancies. Various growth factors, including epidermal growth factor receptor, have been shown to influence pituitary tumor growth and differentiation. To analyze the role of epidermal growth factor receptor in pituitary tumor development, we examined normal pituitaries (n=8), pituitary adenomas (n=158), and pituitary carcinomas (n=7) for expression of epidermal growth factor receptor protein and messenger RNA using tissue microarrays and RT-PCR. We also examined (a) the expression of phospho-epidermal growth factor receptor, the activated form of epidermal growth factor receptor, in pituitary tumors and normal pituitaries by immunohistochemistry and (b) the effects on epidermal growth factor receptor expression of treating pituitary cells (HP75 cell line) with epidermal growth factor. Epidermal growth factor receptor and the phosphorylated variant expression were present in normal pituitary cells. Epidermal growth factor receptor messenger RNA was also detected in normal pituitaries, pituitary adenomas, and carcinomas by in situ hybridization and RT-PCR. Most pituitary adenomas showed expression of epidermal growth factor receptor and the phosphorylated variant. Nonfunctional adenomas showed higher levels of expression of epidermal growth factor receptor (76 vs 34%) and of phospho-epidermal growth factor receptor (26 vs 8%) as compared to functional adenomas. Five of seven pituitary carcinomas showed strong expression of both epidermal growth factor receptor and phospho-epidermal growth factor receptor. When a human pituitary cell line (HP75) was cultured in the presence of epidermal growth factor receptor, there was an increase in the levels of both epidermal growth factor receptor and phospho-epidermal growth factor receptor after 5 h of treatment, thus confirming that epidermal growth factor receptor signaling was active in pituitary tumors. These results indicate that activated epidermal growth factor receptor is expressed in pituitary adenomas and carcinomas. Higher levels in pituitary carcinomas suggest a role in pituitary tumor progression.     

抗人VEGF受体Ⅱ胞外Ⅲ区单克隆抗体的生物学活性研究

目的研究抗KDR单体Ycom1D3抑制VEGF诱导脐静脉内皮细胞（HUVEC）增殖的体外生物学活性。方法采用FACS鉴定Ycom1D3与抗原结合特异性，采用免疫共沉淀测定Ycom1D3阻断VEGF165刺激KDR酪氨酸激酶受体磷酸化作用，并采用[^3H]-Thymidine掺入法、内皮细胞损伤愈合试验和内皮细胞血管腔形成实验进一步确定Ycom1D3抑制VEGF165诱导内皮细胞增殖的中和活性。结果Ycom1D3不但能与HUVEC结合，而且能阻断由VEGF165刺激HUVEC表面KDR酪氨酸激酶受体磷酸化，进而显著抑制VEGF165诱导HUVEC增殖、迁移及体外三维胶原模型上内皮细胞毛细血管样结构的形成。结论Ycom1D3可以通过封闭KDR而抑制VEGF活性，在肿瘤及其它血管新生疾病治疗中具有潜在应用前景。     

Epitope mapping of epidermal growth factor receptor (EGFR) monoclonal antibody and induction of growth-inhibitory polyclonal antibodies by vaccination with EGFR mimotope

One of the proposed approaches in cancer therapy is to induce and direct the patient’s own immune system against cancer cells. In this study, we determined the epitope mapping of the rat anti-human epidermal growth factor receptor (EGFR) monoclonal antibody ICR-62 using a phage display of random peptide library and identified a 12 amino acids peptide, which was recognized as a mimotope. The peptide was synthesized and conjugated to bovine serum albumin (BSA) as carrier protein (P-BSA). We have shown that ICR-62 can react specifically with P-BSA as well as native EGFR. Two rabbits were immunized either by BSA or P-BSA and the rabbits IgGs were purified and examined for binding to the antigens, mimotope and the EGFR protein purified from the EGFR overexpressing A431 cell line. We showed that the rabbit IgG generated against the mimotope is capable of inhibiting the growth of A431 cells by 15%, but does not have any effect on the growth of EGFR-negative MDA-MB-453 cell line in vitro. Our results support the need for further investigations on the potential of vaccination with either mimotope of the EGFR or epitope displayed on the surface of phage particles for use in active immunotherapy of cancer.     

Domain-level antibody epitope mapping through yeast surface display of epidermal growth factor receptor fragments

Individual domains from extracellular proteins are potential reagents for biochemical characterization of ligand/receptor interactions and antibody binding sites. Here, we describe an approach for the identification and characterization of stable protein domains with cell surface display in Saccharomyces cerevesiae, using the epidermal growth factor receptor (EGFR) as a model system. Fragments of the EGFR were successfully expressed on the yeast cell surface. The yeast-displayed EGFR fragments were properly folded, as assayed with conformationally specific EGFR antibodies. Heat denaturation of yeast-displayed EGFR proteins distinguished between linear and conformational antibody epitopes. In addition, EGFR-specific antibodies were categorized based on their ability to compete ligand binding, which has been shown to have therapeutic implications. Overlapping EGFR antibody epitopes were determined based on a fluorescent competitive binding assay. Yeast surface display is a useful method for identifying stable folded protein domains from multidomain extracellular receptors, as well as characterizing antibody binding epitopes, without the need for soluble protein expression and purification.     

Expression of the epidermal growth factor receptor gene in human brain metastases.

Biopsy specimens of human brain metastases were examined for amplification and expression of the proto-oncogene c-erbB1 (located on chromosome 7) encoding the epidermal growth factor receptor (EGFR). Moreover, the tumour DNA was also examined for amplification of other cancer-related genes on this chromosome: the proto-oncogene c-met, the gene for platelet-derived growth factor A-chain, and the gene for plasminogen activator inhibitory type 1. All 18 brain metastases demonstrated positive binding of biotinylated EGF on cryosections. Three out of 18 metastases had amplification of the EGFR gene; the other chromosome-7 genes tested were not amplified. Thus, an increased EGFR gene expression seems to be a general finding in a wide range of carcinomas metastatic to the brain, whereas we found only occasional selective EGFR gene amplifications in single cases.     

Phase I clinical evaluation of a neutralizing monoclonal antibody against epidermal growth factor receptor in advanced brain tumor patients: preliminary study

High levels of growth factors and their receptors have been demonstrated in human tumors. Gliomas and meningiomas are characterized by overexpression of epidermal growth factor receptor (EGF-R). Ior egf/r3, is a neutralizing murine monoclonal antibody (MAb) against EGF-R, and was generated at the Cuban Institute of Oncology. The antibody recognizes EGF-R with high affinity, inhibiting tyrosine kinase activation. A clinical trial was conducted in brain tumor patients to evaluate toxicity, immunogenicity, and clinical benefit of escalating doses of the antibody. Nine patients with histologically confirmed gliomas or meningiomas, who had active or recurrent disease after receiving conventional treatment, received four intravenous doses of ior egf/r3. Total dosages ranged from 160 to 480 mg. As inclusion criteria, radioimmunoscintigraphy with the same MAb labeled with 99mTechnetium (99mTc) was performed. Immune response against the murine antibody was also evaluated. After four doses of ior egf/r3 MAb, no significant toxicity was found, except in one patient who developed a grade 4 allergic adverse event. This reaction was probably related with previous sensitization to the same MAb and the development of human anti-mouse antibodies (HAMA) response. Despite no major objective antitumor responses, eight patients had stable disease on the 6-month evaluation, and two patients remain alive after four years of MAb therapy.