Autoantibody to p185 erbB2/neu oncoprotein by vaccination with xenogenic DNA

 The passive transfer of antibodies and vaccination procedures against p185, the erbB2/neu oncoprotein, are approaches being explored for treatment of human breast cancer. We now report the possibility of using the erbB2/neu gene as an immunogen. This study demonstrates that intramuscular or intradermal injections of rat neuNT full-length DNA into mice generate anti-p185 autoantibodies. Anti-p185 polyclonals were also shown to bind the homologous human receptor ErbB2 and to stain specimens of breast adenocarcinoma from both neu-transgenic mice and humans. Further, in vitro assays demonstrated that anti-p185 IgG (probably dependent on CD4⁺ Th1) were able to inhibit human SKBR3 tumour cell growth and to mediate their lysis by natural killer cells. The continuous presence of circulating neu autoantibodies in mice did not cause any discernible toxic effects on normal tissues expressing low levels of self-antigen, even after 1 year. Received: 29 August 1996 / Accepted: 31 October 1996     

Antibody-induced activation of p185HER2 in the human lung adenocarcinoma cell line Calu-3 requires bivalency

In the present study we utilized two previously described monoclonal antibodies (mAb), and their respective Fab portions, directed against the extracellular domain of p185^HER2, a transmembrane glycoprotein with intrinsic tyrosine kinase activity coded by theHER2/neu oncogene, to study the mechanism of mAb-induced receptor internalization and phosphorylation. Fluorescence scan analysis and direct binding of radiolabelled mAb and their Fab fragments showed that entire MGR2 and MGR3 mAb were reactive with similar binding affinity on two cell lines (Calu-3 and Sk-Br-3) overexpressing the p185^HER2 receptor, and unreactive on unrelated cells. The corresponding Fab fragments were positive on the related cells, but bound with diminished intensity and affinity. Entire MGR2 and MGR3 induced internalization in both Calu-3 and Sk-Br-3 cells, whereas their Fab portions were not internalized. When the bivalency of the MGR2 Fab fragment was artificially reconstituted by incubation with rabbit anti-(mouse IgG), internalization was obtained. Monovalent binding of the entire labelled antibodies, obtained in the presence of a saturating amount of unlabelled antibody, decreased both the rate and the final amount of internalized antibody. Metabolic labelling and immunoblotting experiments showed that incubation with entire MGR3 amplified the basal phosphorylation of the p185^HER2 receptor in Calu-3 and Sk-Br-3 cells, whereas MGR3 Fab decreased the signal. Taken together, our data indicate that antibody-mediated activation of p185^HER2 in Calu-3 and Sk-Br-3 cells occurs through the dimerization of receptor molecules and that bivalency of the activating antibody is mandatory for induction of internalization and phosphorylation of the receptor. Our data support an allosteric model of activation for the p185^HER2 receptor.     

肿瘤中neu基因的过量表达及其抑制

neu基因编码一种和表皮生长因子受体同源的磷酸蛋白,具有酪氨酸激酶的活性.近年来在多种人类肿瘤中发现neu基因的扩增和(或)过量表达.一些蛋白质因子或化学药物可以在转录水平阻遏neu基因的过量表达或者降低其产物p185^neu的酪氨酸激酶活性,抑制具有neu基因过量表达的癌细胞的转移和增殖.     

Rationally designed anti-HER2/neu peptide mimetic disables P185HER2/neu tyrosine kinases in vitro and in vivo

Monoclonal antibodies specific for the p185HER2/neu growth factor receptor represent a significant advance in receptor-based therapy for p185HER2/neu-expressing human cancers. We have used a structure-based approach to develop a small (1.5 kDa) exocyclic anti-HER2/neu peptide mimic (AHNP) functionally similar to an anti-p185HER2/neu monoclonal antibody, 4D5 (Herceptin). The AHNP mimetic specifically binds to p185HER2/neu with high affinity (KD=300 nM). This results in inhibition of proliferation of p185HER2/neu-overexpressing tumor cells, and inhibition of colony formation in vitro and growth of p185HER2/neu-expressing tumors in athymic mice. In addition, the mimetic sensitizes the tumor cells to apoptosis when used in conjunction with ionizing radiation or chemotherapeutic agents. A comparison of the molar quantities of the Herceptin antibody and the AHNP mimetic required for inhibiting cell growth and anchorage-independent growth showed generally similar activities. The structure-based derivation of the AHNP represents a novel strategy for the design of receptor-specific tumor therapies.     

Differential responses of human tumor cell lines to anti-p185HER2 monoclonal antibodies

The HER2 protooncogene encodes a receptor tyrosine kinase, p185^HER2. The overexpression of p185^HER2 has been associated with a worsened prognosis in certain human cancers. In the present work we have screened a variety of different tumor cell lines for p185^HER2 expression using both enzyme-linked immunosorbent and fluorescence-activated cell sorting assays employing murine monoclonal antibodies directed against the extracellular domain of the receptor. Increased levels of p185^HER2 were found in breast (5/9), ovarian (1/6), stomach (2/3) and colorectal (5/16) carcinomas, whereas all kidney and submaxillary adenocarcinoma cell lines tested were negative. Some monoclonal antibodies directed against the extracellular domain of p185^HER2 inhibited growth in monolayer culture of breast and ovarian tumor cell lines overexpressing p185^HER2, but had no effect on the growth of colon or gastric adenocarcinomas expressing increased levels of this receptor. The most potent growth-inhibitory anti-p185^HER2 monoclonal antibody in monolayer culture, designated mumAb 4D5 (a murine IgG1 antibody), was also tested in soft-agar growth assays for activity against p185^HER2-overexpressing tumor cell lines of each type, with similar results. In order to increase the spectrum of tumor types potentially susceptible to monoclonal antibody-mediated anti-p185^HER2 therapies, to decrease potential immunogenicity issues with the use of murine monoclonal antibodies for human therapy, and to provide the potential for antibody-mediated cytotoxic activity, a mouse/human chimeric 4D5 (chmAb 4D5) and a humanized 4D5 (rhu)mAb 4D5 HER2 antibody were constructed. Both engineered antibodies, in combination with human peripheral blood mononuclear cells, elicited antibody-dependent cytotoxic responses in accordance with the level of p185^HER2 expression. Since this cytotoxic activity is independent of sensitivity to mumAb 4D5, the engineered monoclonal antibodies expand the potential target population for antibody-mediated therapy of human cancers characterized by the overexpression of p185^HER2.     

Down-modulation of an oncogene protein product and reversion of the transformed phenotype by monoclonal antibodies

Exposure of neu-oncogene-transformed NIH 3T3 cells to monoclonal antibodies reactive with the neu gene product, p185, results in the rapid and reversible loss of both cell-surface and total cellular p185. Although not directly cytotoxic, monoclonal anti-p185 antibody treatment causes neu-transformed NIH 3T3 cells to revert to a nontransformed phenotype, as determined by anchorage-independent growth. Isotype matched control antibodies of an unrelated specificity do not affect p185 levels or colony formation in soft agar by neu-transformed NIH 3T3 cells. Soft agar colony formation by NIH 3T3 cells transformed by ras oncogenes is not affected by anti-p185 antibody treatment. Anchorage-independent growth of cells from the ethylnitrosourea-induced rat neuroblastoma line in which neu was originally detected by DNA transfection is also inhibited in the presence of anti-p185 monoclonal antibodies. Collectively, these results suggest that p185 is required to maintain transformation induced by the neu oncogene.     

Vaccination with the extracellular domain of p185 neu prevents mammary tumor development in neu transgenic mice

The HER2/neu oncogene product, p185^{HER2/ neu} , is overexpressed on the surface of many human breast cancers. Strains of transgenic mice have been developed that express the rat neu oncogene in mammary epithelial cells and develop spontaneous mammary tumors that overexpress p185 ^neu . This model provides an ideal system for testing interventions to prevent tumor development. In this study, we immunized neu-transgenic mice with a vaccine consisting of the extracellular domain of p185 ^neu (NeuECD). Immunized mice developed Neu-specific humoral immune responses, as measured by circulating anti-Neu antibodies in their sera, and cellular immune responses, as measured by lymphocyte proliferation to NeuECD in vitro. In addition, the subsequent development of mammary tumors was significantly lower in immunized mice than in controls and vaccine treatment was associated with a significant increase in median survival. Received: 10 September 1998 / Accepted: 17 November 1998     

Conformational changes induced by the transforming amino acid substitution in the transmembrane domain of the neu oncogene-encoded p185 protein

Theneu oncogene is frequently found in certain types of human carcinomas and has been shown to be activated in animal models by nitrosourea-induced mutation. The activating mutation in theneu oncogene results in the substitution of a glutamic acid for a valine at position 664 in the transmembrane domain of the encoded protein product of 185 kda (designated p185), which, on the basis of homology studies, is presumed to be a receptor for an as yet unidentified growth factor. It has been proposed that activating amino acid substitutions in this region of p185 lead to a conformational change in the protein which causes signal transduction via an increase in tyrosine kinase activity in the absence of any external signal. Using conformational energy analysis, we have determined the preferred three-dimensional structures for the transmembrane decapeptide (residues 658–667) of the p185 protein with valine and glutamic acid at the critical position 664. The results indicate that the global minimum energy conformation of the decapeptide from the normal protein with Val at position 664 is an -helix with a sharp bend (CD* conformation at residues 664 and 665) in this region, whereas the global minimum conformation for the decapeptide from the mutant transforming protein with Glu at position 664 assumes an all -helical configuration. Furthermore, the second highest energy conformation for the decapeptide from the normal protein is identical to the global minimum energy conformation for the decapeptide from the transforming protein, providing a possible explanation why overexpression of the normal protein also has a transforming effect. These results suggest there may be a normal and a transforming conformation for theneu-encoded p185 proteins which may explain their differences in transforming activity.     

In the FVB/N HER-2/<Emphasis Type="Italic">neu</Emphasis> transgenic mouse both peripheral and central tolerance limit the immune response targeting HER-2/neu induced by <Emphasis Type="Italic">Listeria monocytogenes</Emphasis>-based vaccines

Listeria monocytogenes-based vaccines for HER-2/neu are capable of breaking tolerance in FVB/N rat HER-2/neu transgenic mice. The growth of implanted NT-2 tumors, derived from a spontaneously occurring tumor in the FVB/N HER-2/neu transgenic mouse, was significantly slower in these mice following vaccination with a series of L. monocytogenes-based vaccines for HER-2/neu. Mechanisms of T cell tolerance that exist in these transgenic mice include the absence of functional high avidity anti-HER-2/neu CD8⁺ T cells and the presence of CD4⁺CD25⁺ regulatory T cells. The in vivo depletion of these regulatory T cells resulted in the slowing in growth of tumors even without the treatment of mice with an anti-HER-2/neu vaccine. The average avidities of responsive CD8⁺ T cells to six of the nine epitopes in HER-2/neu we examined, four of which were identified in this study, are shown here to be of a lower average avidity in the transgenic mice versus wild type FVB/N mice. In contrast, the average avidity of CD8⁺ T cells to three epitopes that showed the lowest avidity in the wild-type mice did not differ between wild type and transgenic mice. This study demonstrates the ability of L. monocytogenes-based vaccines to impact upon tolerance to HER-2/neu in FVB/N HER-2/neu transgenic mice and further defines some of the aspects of tolerance in these mice.     

Rat MAbs to the product of the c-erbB-2 proto-oncogene for diagnosis and therapy in breast cancer

The product of the c-erbB-2 protooncogene (p185) is a member of the EGF receptor family of transmembrane tyrosine kinases. Amplification of this gene and overexpression of the product has been observed in adenocarcinomas and has been correlated with poor prognosis in patients with breast and ovarian cancer. The very low levels of expression of p185 by normal adult tissues makes the receptor an almost tumor-specific target. We have prepared rat monoclonal antibodies against five distinct epitopes on the external domain of the c-erbB-2 product overexpressed by the breast cancer line BT474. The antibodies bind to the protein core of p185 and stain specifically the membranes in frozen sections of tumors overexpressing the c-erbB-2 product. Three of the antibodies, ICR12 (epitope A), ICR54, and ICR55 (epitope E), also stain the cell membrane in formalin-fixed, paraffin-embedded sections and bind to p 185 in Western blots. An investigation of the stability of the antigen-antibody complexes indicates that the majority are not readily internalized by SKOV3 cells growing in vitro. Antibodies ICR12 (IgG2a) and ICR55 (IgG2a), which are directed against separate epitopes on the c-erbB-2 p185, are both of high affinity and immunoreactivity (>75%) and localize specifically and stably to xenografted breast and ovarian carcinomas growing in athymic mice when labeled with¹²⁵I (up to 13% injected dose/g, ICR12 and ICR55) or a range of other radionuclides (up to 20% id/g, ICR12). We conclude that these antibodies may be useful as therapeutic vehicles for targeting radionuclides (imaging and therapy) or enzymes for activating prodrugs (ADEPT).     

Intermolecular association of the p185neu protein and EGF receptor modulates EGF receptor function

We have used cross-linking reagents on cell lines expressing both p185neu and EGFR. The lysates of the cells were precipitated with anti-p185neu or anti-EGFR antibodies. These precipitates included a high molecular weight complex that was identified as an EGFR-p185neu heterodimer. Heterodimerization was found to be induced by exposure to EGR. The EGFR of these cells displayed three affinity states for EGF: low (Kd, approximately 10(-9) M), high (Kd, 10(-9) to 10(-10) M), and very high (Kd, 10(-11) M), as determined by Scatchard analyses. Relatively small levels of EGF had a dramatic biological effect on cells expressing very high affinity EGFR. The very high affinity EGFR disappeared after the cells were treated with anti-p185neu monoclonal antibodies that selectively down-regulated p185neu. EGF and TPA had differential effects on down-modulation of the EGFR in cells that express either one or both species of receptor proteins.     

Immunobiology of HER-2/<Emphasis Type="Italic">neu</Emphasis> oncoprotein and its potential application in cancer immunotherapy

HER-2/neu (also known as HER2 or c-erb-B2) is a 185-kDa protein receptor with tyrosine kinase activity and extensive homology to the epidermal growth factor (EGF) receptor. HER-2/neu is expressed in many epithelial tumors and known to be overexpressed in approximately 20–25% of all ovarian and breast cancers, 35–45% of all pancreatic adenocarcinomas, and up to 90% of colorectal carcinomas. HER-2/neu overexpression represents a marker of poor prognosis. HER-2/neu-positive tumor cells are potentially good targets for tumor-reactive cytotoxic T lymphocytes which have been utilized in immunotherapeutic trials. In addition, the humanized monoclonal antibody Herceptin has been tested in several clinical trials and proved to be an effective adjuvant therapy for HER-2/neu-positive breast and ovarian cancers. Vaccinations aiming at generating T-cell responses are being examined in both experimental and clinical trials. Natural immunity at the level of T and B cells has been observed in patients with HER-2/neu-positive tumors confirming the immunogenicity of HER-2/neu and encouraging vaccination trials with HER-2 protein–derived subunits or synthetic peptides. This review summarizes recent data from patients with various types of HER-2/neu–overexpressing cancers carrying different HLA alleles and exhibiting preexistent immunity to HER-2/neu–derived synthetic peptides. It also discusses potential advantages of the various vaccination approaches to immunotherapy targeting the HER-2/neu molecule.Keywords ImmunobiologyHER-2/neu OncoproteinCancer ImmunologyThis work was presented at the first Cancer Immunology and Immunotherapy Summer School, 8–13 September 2003, Ionian Village, Bartholomeio, Peloponnese, Greece.     

Structural insights into the down-regulation of overexpressed p185(her2/neu) protein of transformed cells by the antibody chA21

p185(her2/neu) belongs to the ErbB receptor tyrosine kinase family, which has been associated with human breast, ovarian, and lung cancers. Targeted therapies employing ectodomain-specific p185(her2/neu) monoclonal antibodies (mAbs) have demonstrated clinical efficacy for breast cancer. Our previous studies have shown that p185(her2/neu) mAbs are able to disable the kinase activity of homomeric and heteromeric kinase complexes and induce the conversion of the malignant to normal phenotype. We previously developed a chimeric antibody chA21 that specifically inhibits the growth of p185(her2/neu)-overexpressing cancer cells in vitro and in vivo. Herein, we report the crystal structure of the single-chain Fv of chA21 in complex with an N-terminal fragment of p185(her2/neu), which reveals that chA21 binds a region opposite to the dimerization interface, indicating that chA21 does not directly disrupt the dimerization. In contrast, the bivalent chA21 leads to internalization and down-regulation of p185(her2/neu). We propose a structure-based model in which chA21 cross-links two p185(her2/neu) molecules on separate homo- or heterodimers to form a large oligomer in the cell membrane. This model reveals a mechanism for mAbs to drive the receptors into the internalization/degradation path from the inactive hypophosphorylated tetramers formed dynamically by active dimers during a "physiologic process."     

p185蛋白及其抗体的研究进展

描述了p185在信号转导过程中的功能,讨论了抗p185抗体的蛋白质工程及它们在肿瘤治疗中的应用.     

Resistance ofHER2/neu-overexpressing tumor targets to lymphokine-activated-killer-cell-mediated lysis: evidence for deficiency of binding and post-binding events

HER2/neu-overexpressing tumor cell lines are relatively resistant to lymphokine-activated killer (LAK) cell cytotoxicity when compared toHER2/neu-nonexpressing lines.HER2/neu ⁺ targets were also resistant to binding by LAK large granular lymphocytes (LGL) as shown by visualization at the single-cell level, a target monolayer binding assay and in cold target inhibition experiments.HER2/neu ⁺ LAK-resistant ovarian cell lines demonstrated an absence of ICAM-1 expression while expression of LFA-3, N-CAM, laminin and ₁ integrins was comparable to that ofHER2/neu ^– targets. In contrast, theHER2/neu ⁺ breast cell line, SKBR-3, which was also resistant to lysis and binding by LAK LGL, demonstrated normal expression of ICAM-1. Anti-ICAM-1 antibodies blocked binding and lysis ofHER2/neu ^– carcinoma targets by LAK cells, further supporting the notion that lack of ICAM-1 expression onHER2/neu ⁺ cells contributes to their resistance. The modest binding and lysis ofHER2/neu ⁺ targets by LAK cells was significantly inhibited by anti-LFA-1 antibodies, suggesting the existence of another counter-receptor for LFA-1 onHER2/neu ⁺ targets. The following also supported deficiencies in post-binding events whenHER2/neu ⁺ cells resisted the lytic activity of LAK cells: (a) when the relative resistance to effector cell binding was overcome by exogenous lectin,HER2/neu ⁺ cell lines were still resistant to LAK cytolysis, and (b)HER2/neu ⁺ targets were resistant to perforin-containing granule extracts obtained from the CTLL-R8 cytotoxic lymphocyte cell line. These results indicate that deficiency in effector binding as well as post-binding events contributes to the resistance ofHER2/neu-overexpressing tumor targets to LAK-cell-mediated lysis.Supported by research funds of the Veteran's Administration, the California Institute for Cancer Research and Jonsson Cancer Center core grant CA 16042 funded by NIH     

Colocalization of the p185HER2 oncoprotein and integrin α6β4 in Calu-3 lung carcinoma cells

Anti-p185^HER2 monoclonal antibodies often show intense reactivity with the basement membrane of tumor cells that overexpress the HER2/neu gene product (p185^HER2). To evaluate a possible interaction between p185^HER2 and adhesion molecules or their receptors, the polarity of p185^HER2 was tested in lung carcinoma cell line Calu-3, which overexpresses this protein, in cultures grown as confluent monolayers or as aggregates. MAb immunostaining patterns indicated that p185^HER2 is concentrated on the baso-lateral membrane of cells and that it colocalizes with the integrin α6β4 at the cell-cell junctions where laminin is also found. The same membrane region showed intense reactivity with antiphosphotyrosine antibodies. Furthermore, integrin clustering induced by the specific antibody was accompanied by the clustering of p185^HER2, as indicated by immunoelectron microscopy, and by a subsequent increase in p185^HER2 tyrosine phosphorylation. Treatment with exogenous laminin also resulted in increased basal levels of p185^HER2 phosphorylation. These data suggest a physical interaction between the integrin and the oncoprotein that might be functionally relevant in directly controlling the tyrosine phosphorylation of the catalytic domain of p185^HER2. © 1994 Wiley-Liss, Inc.     

Binding-induced activation of overexpressed p185HER2 is essential in triggering neuronal differentiation of PC12 cells

To determine whether p185^HER2 overexpression per se triggers p185^HER2 cellular signaling or whether an extracellular signal is required, we transfected PC12 cells with the human erbB-2 proto-oncogene, and established a cell line that overexpresses p185^HER2. PC12-HER2 cells, maintained in suspension culture or plated on a collagen layer, showed the same morphology and growth rate as PC12 and PC12 mock-transfected control cells. When treated with monoclonal antibody (MAb) MGr6 or other anti-p185^HER2 MAbs, PC12–HER2 cells specifically underwent neuronal differentiation comparable to that induced by nerve growth factor (NGF), and the differentiation-inducing effect of the MAb was dramatically enhanced by the addition of a second anti-mouse IgG. MAb-induced cell differentiation correlated with p185^HER2 phosphorylation, recruitment of Shc and Grb-2 transducer molecules into complexes, and MAPK phosphorylation. These data indicate the requirement for a specific binding-induced activation of the overexpressed p185^HER2 receptor in inducing PC12 cell differentiation. PC12-HER2 cells represent a suitable system for selection of p185^HER2-activating ligands (peptides, phage-displayed peptides or proteins) or specific inhibitors of its tyrosine kinase activity. J. Cell. Biochem. 67:316–326, 1997. © 1997 Wiley-Liss, Inc.     

Effect of the tyrosine kinase inhibitor lapatinib on CUB-domain containing protein (CDCP1)-mediated breast cancer cell survival and migration

The surface receptor CUB domain-containing protein 1 (CDCP1) is highly expressed in several adenocarcinomas and speculated to participate in anchorage-independent cell survival and cell motility. Tyrosine kinase phosphorylation seems to be crucial for intracellular signaling of CDCP1. Lapatinib, a tyrosine kinase inhibitor (TKI), is approved for treatment of HER-2/neu overexpressing metastatic breast cancer and functions by preventing autophosphorylation following HER-2/neu receptor activation. This study aimed to investigate the effect of CDCP1 expression on anchorage-independent growth and cell motility of breast cancer cells. Moreover, studies were performed to examine if lapatinib provided any beneficial effect on HER-2/neu^(+)/−/CDCP1⁺ breast cancer cell lines. In our studies, we affirmed that CDCP1 prevents cells from undergoing apoptosis when cultured in the absence of cell–substratum anchorage and that migratory and invasive properties of these cells were decreased when CDCP1 was down-regulated. However, only HER-2/neu⁺, but not HER-2/neu^(+)/− cells showed decreased proliferation and invasion and an enhanced level of apoptosis towards loss of anchorage when treated with lapatinib. Therefore, we conclude that CDCP1 might be involved in regulating adhesion and motility of breast cancer cells but that lapatinib has no effect on tyrosine kinases regulating CDCP1. Nonetheless, other TKIs might offer therapeutic approaches for CDCP1-targeted breast cancer therapy and should be studied considering this aspect.     

Interaction of the neu/p185 and EGF receptor tyrosine kinases: Implications for cellular transformation and tumor therapy

Growth factor receptors such as the epidermal growth factor receptor (EGFR) and the p185^c-neu protein serve vital roles in the transduction of differentiation, developmental, or mitogenic signaling within normal cells. Two methods of analysis suggest that the inappropriately high expression of either protein tyrosine kinase promotes malignant transformation. First, data from in vitro experiments indicate that overexpression of either EGFR or p185^c-neu (or the human homolog c-erbB-2) transforms cell-lines. Second, analysis of primary tumors and tumor cell-lines derived from many epithelial tissues (breast, stomach, ovary, and pancreas) show growth factor receptor gene amplification and elevated protein levels. The physical and functional interaction of p185^c-neu and EGFR leads to the formation of a highly active, heterodimeric tyrosine kinase complex which synergistically activates cellular transformation. Anti-receptor antibodies have shown potential utility for the down modulation of these cell-surface proteins and suppression of the malignant phenotype. Design of organic antibody “mimetics” based on the structure of antireceptor antibodies may provide useful therapies and biological reagents to affect growth factor receptor function.     

富含二硫键的膜蛋白p185胞外区在大肠杆菌中的可溶性表达和性质鉴定

Her2/c-erbB-2基因（其产物为膜蛋白p185）是表皮生长因子受体（EGFR）基因家族的一员,在约30％的乳腺癌中发现了其过量表达。为了鉴定抗p185单克隆抗体的抗原表位并进一步研究它们的相互作用,采用PCR的方法从含Her2/c_erbB_2基因的pBabe/erbB_2质粒中扩增了p185胞外区的富含二硫键的第一、二结构域和第四个结构域。产物克隆到pGEX/4T-1载体后,转化大肠杆菌Origami B（DE3）pLysS菌株,用低浓度IPTG进行低温过夜诱导后将菌体压力破碎,SDS-PAGE检测表达上清,得到了可溶性表达的融合有GST的目的蛋白。经ELISA、Western blot等方法鉴定,可溶性表达产物具有完全的抗体结合活性,且当用凝血酶把GST切掉后该活性仍然保留。P185胞外区融合蛋白的成功表达将为二硫键富含类蛋白的表达提供参考;并为将来具有肿瘤细胞生长抑制活性的抗p185单克隆抗体的抗原表位鉴定,以及为EGFR家族受体的结构和功能关系的研究打下基础。