HER-2/neu基因高表达及靶向治疗

HER-2/neu癌基因在许多肿瘤,如乳腺癌、卵巢癌、非小细胞肺癌等肿瘤中高表达,在肿瘤的发生与发展中起重要作用,与肿瘤的转化、转移、复发、预后差、患者生存期缩短有关。HER-2/neu在乳腺癌过度表达率约为20%～30%,编码蛋白P185HER2属生长因子受体家族,抗P185HER2单克隆抗体(Herceptin)作为靶向药物已临床应用治疗HER2/neu高表达乳腺癌。     

Recombinant anti-human HER2/neu IgG3-(GM-CSF) fusion protein retains antigen specificity and cytokine function and demonstrates antitumor activity

Anti-HER2/neu therapy of human HER2/neu-expressing malignancies such as breast cancer has shown only partial success in clinical trials. To expand the clinical potential of this approach, we have genetically engineered an anti-HER2/neu IgG3 fusion protein containing GM-CSF. Anti-HER2/neu IgG3-(GM-CSF) expressed in myeloma cells was correctly assembled and secreted. It was able to target HER2/neu-expressing cells and to support growth of a GM-CSF-dependent murine myeloid cell line, FDC-P1. The Ab fusion protein activated J774.2 macrophage cells so that they exhibit an enhanced cytotoxic activity and was comparable to the parental Ab in its ability to effect Ab-dependent cellular cytotoxicity-mediated tumor cell lysis. Pharmacokinetic studies showed that anti-HER2/neu IgG3-(GM-CSF) is stable in the blood. Interestingly, the half-life of anti-HER2/neu IgG3-(GM-CSF) depended on the injected dose with longer in vivo persistence observed at higher doses. Biodistribution studies showed that anti-HER2/neu IgG3-(GM-CSF) is mainly localized in the spleen. In addition, anti-HER2/neu IgG3-(GM-CSF) was able to target the HER2/neu-expressing murine tumor CT26-HER2/neu and enhance the immune response against the targeted Ag HER2/neu. Anti-HER2/neu IgG3-(GM-CSF) is able to enhance both Th1- and Th2-mediated immune responses and treatment with this Ab fusion protein resulted in significant retardation in the growth of s.c. CT26-HER2/neu tumors. Our results suggest that anti-HER2/neu IgG3-(GM-CSF) fusion protein is useful in the treatment of HER2/neu-expressing tumors.     

In vivo properties of three human HER2/neu-expressing murine cell lines in immunocompetent mice.

BACKGROUND AND PURPOSE: Expression of the HER2/neu proto-oncogene, a receptor-like transmembrane protein expressed at low levels on some normal cells, is markedly increased in a subset of human breast, colon, lung, and ovarian cancers. A humanized HER2/neu antibody has been tested as a therapeutic agent in several clinical trials, with promising results. We have developed a family of anti-HER2/neu fusion proteins. To evaluate the immunologic efficacy of these proteins, it is critical that tumors expressing the target antigen can grow in immunologically intact mice. METHOD: To produce murine tumors expressing human HER2/neu on the surface, CT26, MC38, and EL4 murine cell lines were transduced by use of a retroviral construct containing the cDNA encoding the human HER2/neu gene. RESULTS: Histologic features and kinetics of tumor growth in subcutaneous space of the human HER2/neu-expressing cells were similar to those of the respective parental cell lines. Intravenous inoculation with these cells induced disseminated malignant disease. Flow cytometric and immmunohistochemical analyses of freshly isolated tumors revealed in vivo expression of human HER2/neu. Secretion of antigen was not detected by use of an ELISA. CONCLUSION: Although an antibody response against the human HER2/neu antigen was observed, this response does not affect the growth rate of the HER2/neu-expressing cells. These murine models may be useful tools for evaluation of anti-cancer therapeutic approaches that target human HER2/neu.     

Biodistribution and radioimmunotherapy of human breast cancer xenografts with radiometal-labeled DOTA conjugated anti-HER2/neu antibody 4D5

HER2/neu oncogene encodes a 185 kDa trans-membrane protein which is overexpressed in 20-30% of breast and ovarian cancers and portends a poor prognosis. We have studied the targeting and therapy of this oncoprotein with 4D5, a murine monoclonal antibody which recognizes a distinct epitope on the extracelluar domain of HER2/neu. We conjugated the antibody with an active ester of the macrocyclic chelating agent DOTA, radiolabeled the conjugate with either (111)In or (90)Y, and studied the antibody distribution and therapy, respectively, in athymic mice bearing xenografts of MCF7/HER2/neu, a human breast cancer cell line transfected with the HER2/neu oncogene. For the biodistribution of (111)In-labeled DOTA-4D5, a high specificity of tumor localization (30% ID/g) was seen with a tumor-to-blood ratio of greater than 2 at 48 h postinjection. Compared to a previously published study with (125)I-labeled 4D5 in beige nude mice bearing NIH3T3/HER2/neu xenografts [De Santes et al. (1992) Cancer Res. 52, 1916-1923], (111)In-labeled 4D5 antibody gave superior antibody uptake in tumor (30% ID/g vs 17% ID/g at 48h). In the therapy study, treatment of the nude mice bearing MCF7/HER2/neu xenografts with 100 microCi (3 microg) of (90)Y-labeled DOTA-4D5 caused a 3-fold reduction of tumor growth compared to untreated controls (injected with human serum albumin) in 40 days. Treatment of animals with 100 microCi of nonspecific antibody (90)Y-labeled DOTA-Leu16 (3 microg) had no tumor growth inhibition. Treatment with unlabeled DOTA-4D5 (3 microg) had a slight effect on tumor growth compared to untreated controls. When analyzed at the level of single animals, no effect was seen in seven of nine animals; however, in two of the animals, tumor growth inhibition was observed. Although a cold antibody therapeutic effect was unexpected at this dose level (3 microg), it may be possible that in some animals that 3 microg of antibody of (90)Y-labeled DOTA-4D5 augmented tumor growth reduction. To further explore the effects of cold antibody treatment alone, animals were treated with 100 or 400 microg of unlabeled 4D5 administered in two doses. These animals showed a 1.7-1.8-fold reduction in tumor growth over 28 days, a result less than that obtained with RIT only.     

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."     

Expression of HER2/neu is uncommon in human neuroblastic tumors and is unrelated to tumor progression

Neuroblastic tumors (NT) are the most frequently occurring extracranial solid tumors during childhood. The overall 5-year survival is approximately 20% for patients with metastatic disease. Novel treatments are therefore intensively sought and tumor-targeted immuno- and chemotherapy appear promising. The HER2/neu oncogene, which is highly homologous to the EGF receptor, was initially isolated from rat neuroblastoma cells. HER2/neu over-expression is frequently detected in breast tumors and constitutes an important unfavorable prognostic factor. HER2/neu is a suitable target for antibody-based immunotherapy, as demonstrated by the clinical efficacy of the Herceptin monoclonal antibody (mAb), which reacts with its extracellular domain. Expression of HER2/neu has also been reported to be a negative prognostic factor in a small survey of NT tumors. Here, we have investigated HER2/neu expression in 14 human and 2 murine neuroblastoma (NB) cell lines by flow cytometric analysis and in 93 NT by means of a certified immunohistochemical system. HER2/neu over-expression was found in 2 human cell lines and 11 tumors (14% for both types of samples). No significant association was found between HER2/neu expression and stage, age, sex, ploidy, histological type or subtype. Moreover, log rank test indicated that overall and event-free survival was not significantly different in HER2/neu positive and negative patients. These data suggest that HER2/neu should not be considered as a relevant prognostic factor in NT, and that HER2/neu-based immunotherapy may be feasible only in a minority of NT patients.     

Tyrosine phosphatase inhibition permits analysis of signal transduction complexes in p185HER2/neu-overexpressing human tumor cells.

The HER2/neu gene encodes a receptor tyrosine kinase that is highly homologous to the epidermal growth factor receptor. Overexpression of the receptor in mammary and ovarian carcinoma correlates with poor patient prognosis. To determine how the overexpression of a normal receptor leads to the generation of an oncogenic signal, we compared the patterns of tyrosine phosphorylation in tumor-derived human cell lines expressing high levels of p185HER2/neu. In intact SKBR3 cells, basal phosphorylation of p185HER2/neu was not detected. However, pretreatment of cells with the tyrosine phosphatase inhibitor, sodium orthovanadate, led to the detection of phosphotyrosine on phospholipase C-gamma (PLC-gamma), GTPase-activating protein but not on the RAF-1 kinase. Strikingly, PLC-gamma was detected in a complex which contained multiple tyrosine-phosphorylated polypeptides. This complex was detected only in cytoplasmic fractions and had a distinct composition in different p185HER2/neu-overexpressing cell lines. Although GTPase-activating protein has been found previously in association with proteins of 190 and 62 kDa in fibroblasts, in SKBR3 cells it was found associated with multiple additional tyrosine-phosphorylated polypeptides. These experiments show that SKBR3 cells possess high levels of protein tyrosine phosphatase that can act upon p185HER2/neu. Moreover, they reveal, for the first time, the presence of PLC-gamma and GTPase-activating protein in cytosolic complexes containing a variety of other tyrosine-phosphorylated polypeptides. These observations suggest novel possibilities for the specific definition of receptor-generated signals in tumor cells.     

Characterisation of an engineered trastuzumab IgE antibody and effector cell mechanisms targeting HER2/neu-positive tumour cells

Trastuzumab (Herceptin), a humanized IgG1 antibody raised against the human epidermal growth factor receptor 2 (HER2/neu), is the main antibody in clinical use against breast cancer. Pre-clinical evidence and clinical studies indicate that trastuzumab employs several anti-tumour mechanisms that most likely contribute to enhanced survival of patients with HER2/neu-positive breast carcinomas. New strategies are aimed at improving antibody-based therapeutics like trastuzumab, e.g. by enhancing antibody-mediated effector function mechanisms. Based on our previous findings that a chimaeric ovarian tumour antigen-specific IgE antibody showed greater efficacy in tumour cell killing, compared to the corresponding IgG1 antibody, we have produced an IgE homologue of trastuzumab. Trastuzumab IgE was engineered with the same light- and heavy-chain variable-regions as trastuzumab, but with an epsilon in place of the gamma-1 heavy-chain constant region. We describe the physical characterisation and ligand binding properties of the trastuzumab IgE and elucidate its potential anti-tumour activities in functional assays. Both trastuzumab and trastuzumab IgE can activate monocytic cells to kill tumour cells, but they operate by different mechanisms: trastuzumab functions in antibody-dependent cell-mediated phagocytosis (ADCP), whereas trastuzumab IgE functions in antibody-dependent cell-mediated cytotoxicity (ADCC). Trastuzumab IgE, incubated with mast cells and HER2/neu-expressing tumour cells, triggers mast cell degranulation, recruiting against cancer cells a potent immune response, characteristic of allergic reactions. Finally, in viability assays both antibodies mediate comparable levels of tumour cell growth arrest. These functional characteristics of trastuzumab IgE, some distinct from those of trastuzumab, indicate its potential to complement or improve upon the existing clinical benefits of trastuzumab.     

Apigenin induces apoptosis through proteasomal degradation of HER2/neu in HER2/neu-overexpressing breast cancer cells via the phosphatidylinositol 3-kinase/Akt-dependent pathway

Apigenin is a low toxicity and non-mutagenic phytopolyphenol and protein kinase inhibitor. It exhibits anti-proliferating effects on human breast cancer cells. Here we examined several human breast cancer cell lines having different levels of HER2/neu expression and found that apigenin exhibited potent growth-inhibitory activity in HER2/neu-overexpressing breast cancer cells but was much less effective for those cells expressing basal levels of HER2/neu. Induction of apoptosis was also observed in HER2/neu-overexpressing breast cancer cells in a dose- and time-dependent manner. However, the one or more molecular mechanisms of apigenin-induced apoptosis in HER2/neu-overexpressing breast cancer cells remained to be elucidated. A cell survival pathway involving phosphatidylinositol 3-kinase (PI3K), and Akt is known to play an important role in inhibiting apoptosis in response to HER2/neu-overexpressing breast cancer cells, which prompted us to investigate whether this pathway plays a role in apigenin-induced apoptosis in HER2/neu-overexpressing breast cancer cells. Our results showed that apigenin inhibits Akt function in tumor cells in a complex manner. First, apigenin directly inhibited the PI3K activity while indirectly inhibiting the Akt kinase activity. Second, inhibition of HER2/neu autophosphorylation and transphosphorylation resulting from depleting HER2/neu protein in vivo was also observed. In addition, apigenin inhibited Akt kinase activity by preventing the docking of PI3K to HER2/HER3 heterodimers. Therefore, we proposed that apigenin-induced cellular effects result from loss of HER2/neu and HER3 expression with subsequent inactivation of PI3K and AKT in cells that are dependent on this pathway for cell proliferation and inhibition of apoptosis. This implies that the inhibition of the HER2/HER3 heterodimer function provided an especially effective strategy for blocking the HER2/neu-mediated transformation of breast cancer cells. Our results also demonstrated that apigenin dissociated the complex of HER2/neu and GRP94 that preceded the depletion of HER2/neu. Apigenin-induced degradation of mature HER2/neu involves polyubiquitination of HER2/neu and subsequent hydrolysis by the proteasome.     

Quantitative assessment of HER-2/neu protein concentration in breast cancer by enzyme-linked immunosorbent assay

PURPOSE: The HER-2/neu protein (p185) has become a promising target for antibody therapy in breast cancer. We tested the feasibility of a quantitative approach for HER-2/neu testing based on the analysis of tumor tissue extracts by an enzyme-linked immunosorbent assay (ELISA). EXPERIMENTAL DESIGN: Tumor tissue extracts of primary human breast cancers (n=124) were prepared using a triton-based buffer. HER-2/neu concentration was quantified by ELISA. Paraffin-embedded tissue sections of the same tumors were analyzed by immunohistochemical staining applying the monoclonal HER-2/neu antibody TAB 250 (n=124) and by chromogenic in situ hybridization (CISH) (n=73). RESULTS: Concentrations of p185 in tissue extracts determined by ELISA varied from 1 to 927 ng per mg protein with a median of 25 ng/mg protein, whereas normal breast tissue showed values from 0.4 to 5.5 ng/mg with a median of 2.2 ng/mg (p<0.0001, Mann-Whitney U test). A significant correlation between p185 concentration and immunohistochemical staining was observed (p<0.0001, Kruskal-Wallis test). In addition, p185 concentration measured by ELISA was correlated with the degree of HER-2/neu gene amplification determined by CISH. HER-2/neu-amplified tumors had significantly higher p185 concentrations (median value 181 ng/mg protein) than non-amplified tumors (median value 20 ng/mg; p<0.0001, Mann-Whitney U test). CONCLUSIONS: ELISA-based measurement of HER-2/neu protein concentration in breast cancer tissue extracts is feasible and provides quantitative results for p185 protein concentrations that correlate closely with HER-2/neu immunoscore and gene amplification.     

p185HER2 monoclonal antibody has antiproliferative effects in vitro and sensitizes human breast tumor cells to tumor necrosis factor.

The HER2/c-erbB-2 gene encodes the epidermal growth factor receptorlike human homolog of the rat neu oncogene. Amplification of this gene in primary breast carcinomas has been show to correlate with poor clinical prognosis for certain cancer patients. We show here that a monoclonal antibody directed against the extracellular domain of p185HER2 specifically inhibits the growth of breast tumor-derived cell lines overexpressing the HER2/c-erbB-2 gene product and prevents HER2/c-erbB-2-transformed NIH 3T3 cells from forming colonies in soft agar. Furthermore, resistance to the cytotoxic effect of tumor necrosis factor alpha, which has been shown to be a consequence of HER2/c-erbB-2 overexpression, is significantly reduced in the presence of this antibody.     

Houttuyninum, an active constituent of Chinese herbal medicine, inhibits phosphorylation of HER2/neu receptor tyrosine kinase and the tumor growth of HER2/neu-overexpressing cancer cells

AimsThe overexpression of HER2/neu receptor plays a key role in tumorigenesis and tumor progression. Small molecules targeting HER2/neu have therapeutic value in cancers that overexpress HER2. In this present study, the effect of houttuyninum, a component in the Chinese herbal medicine Houttuynia cordata Thunb, on HER2/neu tyrosine phosphorylation and its in vivo antitumour activity was investigated.Main methodsThe phosphorylation and expression of proteins were determined by Western blot analysis. The MTT assay was employed to examine the inhibition of cell proliferation in vitro. Xenografts were established in nude mice for evaluating the antitumour activity of houttuyninum in vivo.Key findingsHouttuyninum inhibited phosphorylation of HER2 in a dose-dependent manner with an IC50 of 5.52 μg/ml without reducing HER2/neu protein expression in MDA-MB-453 cells. Houttuyninum also inhibited the activation of ERK1/2 and AKT, downstream molecules in the HER2/neu-mediated signal transduction pathway. In contrast, tyrosine phosphorylation of EGFR was unaffected when the concentration of houttuyninum was increased to 40 μg/ml in both A431 cells and MDA-MB-468 cells. Additionally, houttuyninum preferentially inhibited the growth of MDA-MB-453 cells that overexpressed HER2/neu; the MDA-MB-468 cells that overexpress EGFR remained unaffected. Administration of houttuyninum in vivo resulted in a significant reduction of phosphorylated HER2 levels and in tumor volumes of the BT474 and N87 xenografts, which both overexpress HER2/neu.SignificanceOur findings showed that houttuyninum can inhibit the HER2/neu signalling pathway and the tumor growth of cancer cells that overexpress HER2/neu. This drug may provide therapeutic value in the treatment of cancers that involve overexpression of HER2/neu.     

基质金属蛋白酶-2和p185HER-2/neu蛋白在卵巢癌中的研究进展

基质金属蛋白酶-2(Matrix Metalloproteinase-2,MMP-2)是基质金属蛋白酶家族的重要成员,能降解明胶蛋白和Ⅳ型、V型胶原,在细胞外基质的降解过程中起着关键作用,能够促进肿瘤细胞发生侵袭和转移。p185HER-2/neu蛋白是一种相对分子质量185×103的跨膜糖蛋白,由HER-2/neu基因编码,属于酪氨酸激酶受体家族,p185HER-2/neu蛋白在人类多种癌症中存在扩增及过量表达,并与肿瘤的侵袭性表型及生存期短密切相关。就基质金属蛋白酶-2和p185HER-2/neu蛋白的生物学特性,与卵巢癌侵袭转移和预后的关系及MMP-2和p185HER-2/neu蛋白的研究情况等予以综述。     

Epidermal growth factor receptor coexpression modulates susceptibility to Herceptin in HER2/neu overexpressing breast cancer cells via specific erbB-receptor interaction and activation

BACKGROUND: Growth factors and Herceptin specifically and differentially modulate cell proliferation of tumor cells. However, the mechanism of action on erbB-receptor level is incompletely understood. We evaluated Herceptin's capacity to modulate erbB-receptor activation and interaction on the cell surface level and thereby potentially impair cell proliferation of HER2/neu (c-erbB2) overexpressing breast cancer cells, both in the presence and absence of relevant growth factors. METHODS: BT474 and SK-BR-3 breast cancer cell lines were treated with Epidermal Growth Factor (EGF), Heregulin, and with Herceptin in different combinations. Kinetics of cell proliferation were evaluated flow cytometrically based on BrdU-labeling. Fluorescence Resonance Energy Transfer, ELISAs and phosphorylation site specific Western Blotting was performed to investigate erbB-receptor interaction and activation. RESULTS: EGF induced EGFR/EGFR and EGFR/c-erbB2 interactions correlate with stimulation of cell proliferation in BT474 cells. Both homo- and heterodimerization are considerably less pronounced in SK-BR-3 cells and heterointeraction is additionally reduced by EGF treatment, causing inhibition of cell proliferation. Heregulin stimulates cell proliferation extensively in both cell lines. Herceptin drives BT474 cells more efficiently into quiescence than it does with SK-BR-3 cells and thereby blocks cell cycle progress. In SK-BR-3 Herceptin treatment causes c-erbB2 phosphorylation of Y877 and Y1248, EGF induces Y877 and Y1112 phosphorylation. The Y1112 phosphorylation site, activated by EGF in SK-BR-3 cell, is bypassed in BT474. In addition the inhibitory capacity of Herceptin on BT474 and SK-BR-3 cell proliferation depends on the presence and absence of growth factors to a various extent. CONCLUSION: The growth inhibitory effect of Herceptin on c-erbB2 overexpressing breast cancer cells is considerably modulated by EGFR coexpression and consequently EGFR/c-erbB2 homo- and heterointeractions, as well as the presence or absence of growth factors. C-erbB2 overexpression alone is insufficient to predict the impact of growth factors and antibodies on cell proliferation. The optimization and specification of therapeutic approaches based on erbB-receptor targeting requires to account for EGFR coexpression as well as the potential presence of erbB-receptor relevant growth factors.     

Regulation of phosphorylation of the c-erbB-2/HER2 gene product by a monoclonal antibody and serum growth factor(s) in human mammary carcinoma cells.

Monoclonal antibody (MAb) 4D5 was used to analyze the phosphorylation of p185HER2, the gene product of c-erbB-2/HER2, in SK-BR-3 cells. Culture in the continuous presence of 4D5 reduced the in vivo steady-state levels of p185HER2 phosphorylation by 80% in a dose-dependent manner, suggesting that MAb 4D5 may have interfered with the activation of phosphorylation of p185HER2. The observed MAb-mediated reduction of p185HER2 phosphorylation could not be completely accounted for by down-regulation. When cultures were grown under serum-free conditions, the steady-state levels of p185HER2 phosphorylation were reduced by 56%, and addition of 4D5 further inhibited phosphorylation to 20% of steady-state levels. With continuous exposure to increasing concentrations of newborn calf serum in these cultures, there was a linear increase in tyrosine-specific phosphorylation of p185HER2, reaching a 5.4-fold increase with 10% newborn calf serum. Phosphorylation of p185HER2 in the presence of newborn calf serum was not attributable to stimulation of the epidermal growth factor receptor by epidermal growth factor or by transforming growth factor-alpha. Extension of these observations to two other mammary carcinoma cell lines. MDA-MB-453 and BT-474, also demonstrated a significant capacity of serum to induce p185HER2 phosphorylation. The demonstration of antibody-mediated partial inhibition of phosphorylation under serum-free conditions suggests that mammary carcinoma cells may also produce and secrete a factor or factors which may activate p185HER2. Our observation that growth-inhibitory MAb 4D5 is able to reduce the phosphorylation of p185HER2 by newborn calf serum and by a cellular-derived factor(s) suggests the existence of a growth factor(s) which uses phosphorylation of p185HER2 as a signal transduction pathway to regulate cell proliferation.     

Immunoprevention of mammary carcinoma in HER-2/neu transgenic mice is IFN-gamma and B cell dependent

A vaccine combining IL-12 and allogeneic mammary carcinoma cells expressing p185(neu) completely prevents tumor onset in HER-2/neu transgenic BALB/c mice (NeuT mice). The immune protection elicited was independent from CTL activity. We now formally prove that tumor prevention is mainly based on the production of anti-p185(neu) Abs. In the present studies, NeuT mice were crossed with knockout mice lacking IFN-gamma production (IFN-gamma(-/-)) or with B cell-deficient mice (microMT). Vaccination did not protect NeuT-IFN-gamma(-/-) mice, thus confirming a central role of IFN-gamma. The block of Ab production in NeuT-microMT mice was incomplete. About one third of NeuT-microMT mice failed to produce Abs and displayed a rapid tumor onset. By contrast, those NeuT-microMT mice that responded to the vaccine with a robust production of anti-p185(neu) Ab displayed a markedly delayed tumor onset. In these NeuT-microMT mice, the vaccine induced a lower level of IgG2a and IgG3 and a higher level of IgG2b than in NeuT mice. Moreover, NeuT-microMT mice failed to produce anti-MHC class I Abs in response to allogeneic H-2(q) molecules present in the cell vaccine. These findings show that inhibition of HER-2/neu carcinogenesis depends on cytokines and specific Abs, and that a highly effective vaccine can rescue Ab production even in B cell-deficient mice.     

Biological studies and potential therapeutic applications of monoclonal antibodies and small molecules reactive with theneu/c-erbB-2 protein

The overexpression of the growth factor receptor p185 ^neu/c-erbB-2 has been observed in a number of human adenocarcinomas and is mechanistically linked to neoplastic growth. Monoclonal antibodies raised against extracellular domains of the p185 ^neu/c-erbB-2 receptor oncoprotein have been utilized to inhibit the pathway ofneu-induced tumor development. Our laboratory has demonstrated a direct effect of anti-p185 ^neu/c-erbB2 antibodies which requires receptor ligation. This induced aggregation causes the downmodulation of cell-surface expression and eventual degradation of p185 ^neu/c-erbB-2 protein. In cells transformed by theneu oncogene, the result of antibody-induced p185 ^neu/c-erbB-2 receptor modulation is the reversion of the malignant pheno-type. We are exploiting the direct efficacy of this monoclonal antibody by developing small molecules (peptides and organic mimietics) based on anti-p185 ^neu/c-erbB-2 antibody structure that can mediate similar receptor binding and biological effects.