Humanized anti-HER2 Monoclonal Antibody Research Articles

Novel targeted therapies in inflammatory breast cancer

Inflammatory breast cancer (IBC) accounts for 1% to 5% of all breast cancer cases. Its aggressive biology is characterized by rapid disease progression and poor prognosis. To improve and standardize therapy for IBC, development of novel therapeutics to molecular targets of IBC is key. Trastuzumab treatment, an anti-HER2 humanized monoclonal antibody, has been demonstrated to improve the 3-year survival rate of patients with IBC (70.1% vs 53.3%; P = .0007). Another chemotherapeutic drug, lapatinib, a dual tyrosine inhibitor of epidermal growth factor (EGFR) and HER2 signaling, in combination with capecitabine demonstrated encouraging results, with a 51% decrease in disease progression. Further investigation is needed to confirm the efficacy of lapatinib.

Abstract 3779: Combination chemoprevention of hepatocellular carcinoma using acyclic retinoid

Abstract Background and aims: A malfunction of retinoid X receptor-α (RXRα) due to phosphorylation by the Ras/MAPK signaling pathway is associated with the development of hepatocellular carcinoma (HCC). Acyclic retinoid (ACR: the same material as NIK-333), which has been clinically shown to reduce the incidence of a post-therapeutic recurrence of HCC, can inhibit both the Ras activity and phosphorylation of the ERK and RXRα proteins. The effects of a combination of ACR plus other agents on the inhibition of cell growth in human HCC cells were herein examined to develop a more effective strategy for the prevention of HCC. Methods and results: The combination of ACR plus vitamin K2, which inhibits the phosphorylation of the RXRα protein through the inhibition of Ras/MAPK activation, synergistically induced apoptosis and inhibited the growth of human HCC cells without affecting the growth of normal human hepatocytes. ACR and trastuzumab, the humanized anti-HER2 monoclonal antibody, also synergistically inhibited the activation of HER2, a member of receptor tyrosine kinase (RTK), and its downstream signaling pathways, thus subsequently inhibiting the phosphorylation of RXRα and the growth of HCC cells. ACR in combination with valproic acid, a histone deacetylase inhibitor, also synergistically inhibited the growth of human HCC cells and this was associated with the inhibition of phosphorylation of the RXRα and Akt proteins by valproic acid. Conclusion: The combination of ACR plus a specific agent that targets RTK and its downstream signaling pathways, including the Ras/MAPK and PI3K/Akt pathways, may be able to inhibit the phosphorylation of RXRα and it may therefore be a promising strategy to prevent the development of HCC. ACR, which targets phosphorylated RXRα, may thus play a critical role in preventing the development of HCC when it is used either alone or in combination with other agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3779.

Abstract 5226: Potential of PET imaging to predict the response to anti-HER2 therapy in breast cancer

Abstract Overexpression of HER2 is associated with resistance to standard therapies and, consequently, poor prognosis. The most successful of HER2-targeted therapies, and the only one approved by the FDA, uses trastuzumab (Herceptin) - a recombinant humanized anti-HER2 monoclonal antibody. Currently, alteration of gross tumor volume is used to assess the response to trastuzumab. However, this approach provides only a late indicator of response. The ex vivo assays of tissue samples are potentially valuable, but their procurement through biopsies is invasive and might be biased by tumor heterogeneity. We studied the feasibility of PET imaging to quantify changes in HER2 expression and to predict the response to trastuzumab in breast cancer xenograft model using 18F-ZHER2-Affibody molecule as a tracer. We administered the tracer to mice bearing subcutaneous BT474 tumors, which display very high HER2 expression. The control and treatment groups consisted of 15-20 animals each. The tumor volume was in 100-250 mm3 range. The tumor size was monitored twice weekly using caliper measurements. Trastuzumab (50 mg/kg loading dose, 25 mg/kg maintenance dose) or saline (control) were given i.p. twice a week for a total of 5 doses. Animals were scanned before the treatment, 48 h and two weeks after the beginning of therapy. Immediately after the last scan, animals were euthanized and pieces of tumors were fixed and/or frozen for further analysis. The HER2 expression and activation of HER2 signaling pathways in the tumor samples were assessed by Western blot, ICH and ELISA. Quantitative analysis of PET images indicated decrease of HER2 expression, associated with significant tumor regression (>30% volume reduction), in 12 of 20 treated tumors. However, only negligible changes in HER2 expression and stable disease were found in the remaining treated mice. No changes in HER2 expression and intensive growth of the tumors (increase more than 100% of tumor volume) were observed in the control group over the 2 weeks of therapy. These results were confirmed by ex vivo analysis showing dramatic decrease of total HER2 level in responding tumors. Despite the reduction of HER2 levels, there was persistent HER2 phosphorylation in all tumors. As previously shown, it might be due to activation of alternative HER receptors as a result of ADAM17-mediated autocrine ligand release. Tissue analysis showed a trend for trastuzumab-treated xenografts to have higher levels of ADAM17 and heregulin compared to the untreated mice. Other mechanisms that might account for the observed difference between the responders and the non-responders are currently investigated. Our results suggest that monitoring of changes in HER2 expression after five doses of trastuzumab allows predicting the tumor response and identifying mice that would not fully benefit from the treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5226.

Abstract CN3-1: Detection, clinical relevance, and molecular characteristics of circulating tumor cells

Abstract Early spread of tumor cells is usually undetected even by high-resolution imaging technologies, which prevents effective early intervention. However, sensitive immunocytochemical and molecular assays have become available and enable now the specific detection of 'occult' metastatic tumor cells even at the single-cell stage. These technologies provide the potential to track systemic tumor cell dissemination in cancer patients. Disseminated tumor cells (DTC) have been frequently detected in the bone marrow (BM) of patients with diverse types of solid tumors, suggesting that the BM might be a preferred reservoir for blood-borne DTC. Whether DTC use this environment as niche to persist in a dormant state over many years before they disseminate into other organs or grow out in the BM into overt metastases is subject of current investigations. To understand this stage of dormancy and the conditions enabling DTC to reactivate growth as well as to identify the founder cells of overt metastases (“metastatic stem cells”) are some of the most important and challenging research areas. The observed correlation between DTC in BM and local relapse in breast cancer support the recent experimental findings in mice that these cells might re-circulate back to their primary organ site. If BM is a particular reservoir for DTC, drugs targeting the BM-tumor interaction (e.g., bisphosphonates or antibodies to the RANK ligand) may be efficient to prevent metastatic or even local relapse. In clinical practice, peripheral blood drawings are more acceptable than BM aspirations in solid tumor patients. Many research groups are therefore assessing the clinical value of circulating tumor cells (CTC) for therapy monitoring, which has provided significant prognostic information in metastatic breast cancer, and seems to be superior over conventional imaging methods for response evaluation. The clinical utility of these findings are now being prospectively addressed in a randomized trial SWOG S0500 led by the Southwest Oncology group (www.cancer.gov/clinicaltrials/SWOG-S0500). While the prognostic significance of CTC could be demonstrated for metastatic breast, colorectal or prostate cancer patients, studies on the impact of CTC in early stage cancer patients are still ongoing. Thus far, encouraging results on monitoring of CTC during primary systemic or adjuvant chemotherapy in breast cancer patients were obtained. Phenotypical and molecular characterization of DTC/CTC will be essential to identify new diagnostic and therapeutic targets and contribute to more “tailored” and personalized anti-metastatic therapies (www.dismal-project.eu). The identification of therapeutically relevant targets may contribute to a better selection of cancer patients for targeted therapies. In breast cancer, HER2 has become the most prominent target for biological therapy with a humanized anti-HER2 monoclonal antibody. Currently, all patients are stratified to trastuzumab therapy by primary tumor analysis only. However, recent reports have shown that HER2-positive DTC/CTC can be detected also in patients with HER2-negative primary tumors (Pantel et al NRC 2008), suggesting that additional patients could benefit from HER2-directed therapies. Ongoing clinical studies will reveal whether the HER2 status of CTC may predict response to trastuzumab or other HER2-directed therapies. These results may have also an important impact on the future design of clinical trials testing other promising targets expressed on micrometastatic cancer cells. In conclusion, information obtained from DTC/CTC analyses might be useful to obtain prognostic information, monitor the efficacy of anti-cancer therapies, identify therapeutic targets on metastatic cells and provide new insights into the biology of metastatic progression in cancer patients. Citation Information: Clin Cancer Res 2010;16(7 Suppl):CN3-1

Lyophilized HER2-specific PEGylated immunoliposomes for active siRNA gene silencing

The development of a tumor-specific immunoliposome delivering small interfering RNA (siRNA) represents a practical way in cancer gene therapy. In this study, we developed PEGylated 3β-[N-(N′, N′-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol)/dioleoylphosphatidyl ethanolamine (DOPE) immunoliposomes conjugated with the Fab' of recombinant humanized anti-HER2 monoclonal antibody (PIL) for siRNA delivery. The results demonstrated that the lyophilized PIL (LPIL) prepared by the lyophilization/rehydration method possessed a significantly enhanced HER1 gene, a model target, silencing ability compared with PIL in HER2-overexpressing SK-BR3 cells. Among a series of LPIL with different PEGylation degree, LPIL containing 2.5%PEG (2.5%PEG LPIL) showed the best HER1 gene silencing activity. Confocal microscope studies demonstrated that 2.5%PEG LPIL could specifically bind to SK-BR3 cells and were sequentially internalized into them. Using RhoA as a cancer therapeutic target, 2.5%PEG LPIL entrapping anti-RhoA siRNA could specifically silence RhoA expression and inhibit cell invasion in SK-BR3 cells. In conclusion, these finding demonstrated the potential use of 2.5%PEG LPIL in specifically delivering siRNA to HER2-overexpressing cancers.

Modulation of HER3 Is a Marker of Dynamic Cell Signaling in Ovarian Cancer: Implications for Pertuzumab Sensitivity

This study was designed to evaluate the expression of HER receptors as a marker of sensitivity to the humanized anti-HER2 monoclonal antibody pertuzumab in ovarian cancer cells. In a recent clinical trial, low levels of HER3 mRNA have been shown to associate with pertuzumab response when combined with gemcitabine. We sought to define how pertuzumab modulated HER expression levels in ovarian cancer using cell line models to better understand differential and dynamic receptor expression in therapeutic response. Changes in HER3 mRNA expression were also assessed in pertuzumab-treated xenografts. HER3 mRNA and, to a lesser extent, HER2, were down-regulated after stimulation both with heregulin-beta1 and epidermal growth factor in a range of ovarian cancer cell lines either growth sensitive or growth resistant to pertuzumab. Pertuzumab reversed this down-regulation and the magnitude of the reversal correlated with pertuzumab sensitivity. The change in HER3 mRNA expression correlated inversely to how much the extracellular signal-regulated kinase and phosphoinositide 3-kinase pathways were dynamically activated with stimulation. Finally, up-regulation of HER3 mRNA was found in cancer xenografts treated with pertuzumab. We conclude that HER3 mRNA is down-regulated by both heregulin-beta1 and epidermal growth factor activation. This suggests that in some tumors, low HER3 mRNA expression is driven by, or dependent on, growth factor. HER3 mRNA expression is effectively reversed in pertuzumab-sensitive tumors. These data are consistent with low HER3 mRNA identifying a pertuzumab-sensitive phenotype.

HER2 status testing by immunohistochemical and fFluorescence in situ hybridization in China

e22233 Background: HER2 gene overexpression is associated with aggressive breast cancer and poor clinical prognosis. Humanized anti-HER2 monoclonal antibody trastuzumab, which is targeted HER2 protein has showed to improve overall survival in patients with HER2-positive breast cancer in both the metastatic and adjuvant settings. There are some differences in HER2 positive rate among difference reports in China. This study tested HER2 status by immunohistochemistry(IHC) and fluorescence in situ hybridization (FISH) and compared HER2 testing at central and regional laboratories in China. Methods: Assessment of HER2 status was performed by FISH using the HercepTeast kit at central laboratory and by IHC using commercial available anti-HER2 probe in formalin-fixed and paraffin-embedded tissue section of 280 breast cancer samples. IHC HER2 testing was performed on 149 samples in the central laboratory. IHC HER2 testing was performed on 80 samples at both central laboratory and regional laboratory. Results: 280 samples were tested 373 times testing by IHC and FISH. The results were showed in table 1 . 80 samples was tested by IHC at central and regional laboratory and testing results of 36.4% samples were accordant (K=0.038). 94.1% IHC3+ at central laboratory were HER2 FISH positive and 83.3% IHC 3+ at regional laboratory were HER2 FISH positive. 86.7% IHC 2+ at central laboratory were HER2 FISH positive and 62.7% IHC 2+ at regional laboratory were HER2 FISH positive. 17 samples were observed HER2 FISH positive in the 27 IHC 0/1+ tested at regional laboratoty. So good correlation was obsearved between FISH HER2 status and IHC results from central laboratory but not from regional laboratory. Conclusions: This study emphasized the important of accurate HER2 testing. HER2 FISH test should be performed for the IHC 2+ samples. Even HER2 FISH test maybe performed for IHC 0/1 sample according to clinical characteristics in China in order to make the patients have targeted therapy chance. [Table: see text] No significant financial relationships to disclose.

Tumor-targeted PE38KDEL delivery via PEGylated anti-HER2 immunoliposomes

We previously reported the development of PE38KDEL-loaded anti-HER2 poly(lactic-co-glycolic acid) (PLGA) nanoparticles that bind and internalize in HER2-overexpressing breast cancer cells, enabling potent anti-tumor activity. To overcome the problems associated with the short half-lives of this drug delivery system, we have constructed PE38KDEL-loaded anti-HER2 PEGylated liposomes (PE-HER-liposomes). PE-HER-liposomes were constructed with Fab′ of recombinant humanized anti-HER2 monoclonal antibody (anti-HER2 Fab′) covalently linked to PEGylated liposomes containing PE38KDEL (PE-liposomes). We attached anti-HER2 Fab′ to the terminus of PEG (polyethylene glycol) on PEGylated liposomes. Incorporation of pyridylthiopropionoylamino-PEG-distearoylphosphatidylethanolamine (PDP-PEG-DSPE) into PEGylated liposomes followed by mild thiolysis of the PDP groups resulted in the formation of reactive thiol groups at the periphery of the liposomes. Efficient attachment of maleimide-derivatized anti-HER2 Fab′ took place under mild conditions. The characterization of PE-HER-liposomes, such as particle size, was evaluated by dynamic light-scattering detector. The Micro BCA method was used to determine the encapsulation efficiency of PE38KDEL and the quantity of conjugated Fab′. Flow cytometry and confocal microscopy showed that PE-HER-liposomes possessed receptor-specific binding and internalization for HER2-overexpressing SK-BR3 cells. Remarkably, PE-HER-liposomes were more cytotoxic than non-targeted PE-liposomes in HER2-overexpressing breast cancer cells. In conclusion, PE-HER-liposomes could serve as a promising therapeutic candidate for the treatment of HER2-overexpressing breast cancers.

Imaging of HER-2 Overexpression in Tumors for Guiding Therapy

Human epidermal growth factor receptor type 2 (HER2) is a transmembrane tyrosine kinase receptor, which is overexpressed in a large fraction of breast, ovarian, urinary bladder and a number of other carcinomas. Overexpression of HER2 is associated with poor prognosis. Treatment of patients with HER2-expressing breast cancer with a humanized anti-HER2 monoclonal antibody trastuzumab has resulted in improved survival. Several kinds of other anti-HER2 therapies are under development. Radionuclide molecular imaging of HER2 expression may influence patient management by selecting patients, who would benefit form anti-HER2 therapy. Other applications, such as therapy response monitoring and follow-up are also possible. In this case, the use of radionuclide imaging may overcome problems associated with biopsies, including sampling errors and discordance of expression between primary tumors and metastases. Important preconditions for development of a successful tracer for radionuclide imaging are high affinity of a targeting agent and suitable chemistry of labeling. The paper reviews information concerning major classes of HER2-targeting agents, including full-length monoclonal antibodies, their enzymatically produced fragments, engineered immunoglobulin based tracers, and alternative high affinity binders. Available information suggests that Affibody molecules or other small non-immunoglobulin based tracers have the best potential for development of high-contrast imaging agents for visualization of HER2 in vivo.

PE38KDEL-loaded anti-HER2 nanoparticles inhibit breast tumor progression with reduced toxicity and immunogenicity

The clinical use of Pseudomonas exotoxin A (PE)-based immunotoxins is limited by the toxicity and immunogenicity of PE. To overcome the limitations, we have developed PE38KDEL-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles conjugated with Fab' fragments of a humanized anti-HER2 monoclonal antibody (rhuMAbHER2). The PE38KDEL-loaded nanoparticles-anti-HER2 Fab' bioconjugates (PE-NP-HER) were constructed modularly with Fab' fragments of rhuMAbHER2 covalently linked to PLGA nanoparticles containing PE38KDEL. Compared with nontargeted nanoparticles that lack anti-HER2 Fab', PE-NP-HER specifically bound to and were sequentially internalized into HER2 overexpressing breast cancer cells, which result in significant cytotoxicity in vitro. In HER2 overexpressing tumor xenograft model system, administration of PE-NP-HER showed a superior efficacy in inhibiting tumor growth compared with PE-HER referring to PE38KDEL conjugated directly to rhuMAbHER2. Moreover, PE-NP-HER was well tolerated in mice with a higher LD(50) (LD(50) of 6.86 +/- 0.47 mg/kg vs. 2.21 +/- 0.32 mg/kg for PE-NP-HER vs. PE-HER (mean +/- SD); n = 3), and had no influence on the plasma level of plasma alanine aminotransferase (ALT) of animals when injected at a dose of 1 mg/kg where PE-HER caused significant increase of serum ALT in the treated mice. Notably, PE-NP-HER was of low immunogenicity in development of anti-PE38KDEL neutralizing antibodies and was less susceptible to inactivation by anti-PE38KDEL antibodies compared with PE-HER. This novel bioconjugate, PE-NP-HER, may represent a useful strategy for cancer treatment.

Synergistic Growth Inhibition by 9-cis-Retinoic Acid Plus Trastuzumab in Human Hepatocellular Carcinoma Cells

A malfunction of retinoid X receptor-alpha (RXRalpha) due to phosphorylation by the Ras/mitogen-activated protein kinase signaling pathway is associated with the development of hepatocellular carcinoma (HCC). The humanized anti-HER2 monoclonal antibody trastuzumab inhibits the activation of HER2 and its multiple downstream signaling pathways, including the Ras/mitogen-activated protein kinase pathway. In this study, the effects of phosphorylation of RXRalpha on the ability of RXRalpha ligand 9-cis-retinoic acid (9cRA) and trastuzumab to inhibit growth of HCC cells was examined. The effects of a combination of 9cRA plus trastuzumab on the inhibition of cell growth in HLF human HCC cells which express constitutive activation of HER2 protein were examined. The combination of 9cRA plus trastuzumab synergistically inhibited the growth of HLF cells without affecting the growth of Hc normal human hepatocytes. Combined treatment with these agents acted synergistically to induce apoptosis in HLF cells. The treatment of HLF cells with trastuzumab alone inhibited the phosphorylation of HER2, RXRalpha, ERK, Akt, and Stat3 proteins and these effects were enhanced when the cells were cotreated with 9cRA. Reporter assays indicated that the combination of 9cRA plus trastuzumab markedly increased both the retinoic acid responsive element and retinoid X responsive element promoter activities in HLF cells. 9cRA and trastuzumab cooperatively inhibit the activation of HER2 and its downstream signaling pathways, subsequently inhibiting the phosphorylation of RXRalpha and the growth of HCC cells. This combination might therefore be effective for the chemoprevention and chemotherapy of HCC.

Preparation and characterization of PE38KDEL-loaded anti-HER2 nanoparticles for targeted cancer therapy

The clinical use of immunotoxins is severely limited by nonspecific toxicity. To overcome this limitation, PE38KDEL was used as a model protein toxin to prepare PE38KDEL-loaded poly(lactic-co-glycolic acid) (PLGA) antibody modified nanoparticles (NPs), which were covalently conjugated with Fab' fragments of a humanized anti-HER2 monoclonal antibody (rhuMAbHER2) by a two-step carbodiimide method. The characterization of the PE38KDEL-loaded nanoparticles-anti-Fab' bioconjugates (PE-NPs-HER), such as particle size, zeta potential and morphology, were evaluated by dynamic light-scattering detector and transmission electron microscope (TEM). Micro BCA assay was used to determine the drug encapsulation efficiency and the quantity of Fab' conjugated with NPs. The binding affinity and internalization efficiency of PE-NPs-HER were demonstrated by flow cytometry and laser-scanning confocal microscopy. In comparison with PE38KDEL-loaded nanoparticles (PE-NPs) that lack anti-HER2 Fab', PE-NPs-HER had superior in vitro cytotoxicity against HER2-overexpressing breast cancer cell lines. Progressively, PE-NPs-HER has superior protective antitumor activity in HER2-overexpressing tumor-bearing mice than the control immunotoxin PE-HER constructed by chemically coupling PE38KDEL to rhuMAbHER2. Most strikingly, in developed HER2-overexpressing tumor xenograft model, administration of PE-NPs-HER (0.9 mg/kg) showed a much better therapeutic efficacy in inhibiting tumor growth compared with PE-HER and other controls: final mean tumor load was 13 ± 6 mm 3 (mean ± SD; n = 8, significantly smaller than all other groups by ANOVA at 95% confidence interval). In addition, PE-NPs-HER were well tolerated in mice with a much higher MTD (maximally tolerated dose) than PE-HER (2.92 mg/kg vs. 0.92 mg/kg), indicating the systemic toxicity of PE38KDEL was dramatically reduced by PLGA encapsulation. Thus, the bioconjugates PE-NPs-HER may represent a potentially useful strategy for cancer therapy.

Tumor Protection Following Vaccination With Low Doses of Lentivirally Transduced DCs Expressing the Self-antigen erbB2

Gene therapy strategies may accelerate the development of prophylactic immunotherapy against cancer. We synthesized a lentiviral (LV) vector encoding a kinase-deficient form of erbB2 (erbB2tr) to transduce murine dendritic cells (DCs) efficiently. Murine erbB2 models a clinically relevant tumor-associated self-antigen; its human homolog (HER-2/neu) is overexpressed in breast cancer and in 80% of metastatic prostate cancers. Following one infection, approximately 47% of DCs overexpressed erbB2tr. To determine whether low doses of transduced DCs could protect mice from prostate cancer cells, we performed prime/boost vaccinations with 2 x 10(3) or 2 x 10(5) erbB2tr-transduced DCs. Six weeks after vaccination, mice were simultaneously bilaterally challenged with the aggressive RM-1 prostate cancer cell line and an erbB2tr-expressing variant (RM-1-erbB2tr). Whereas control mice developed both tumors, all recipients of 2 x 10(5) erbB2tr-transduced DCs developed only wild-type RM-1 tumors. One-third of mice vaccinated with just 2 x 10(3) erbB2tr-transduced DCs also demonstrated erbB2tr-specific tumor protection. Protection against RM-1-erbB2tr tumors was associated with sustained levels of anti-erbB2tr antibody production and also correlated with erbB2tr-specific Th1 cytokine secretion. Depletion of CD4(+), CD8(+), or natural killer (NK) cells prior to tumor challenge underscored their role in mediating tumor protection. We conclude that administration of DCs expressing a self-antigen through efficient LV-based gene transfer activates cellular and humoral immunity, protecting host animals against specific tumor challenge.

Long-Term Cardiac Tolerability of Trastuzumab

Trastuzumab (Herceptin), a humanized anti-HER2 monoclonal antibody, is highly effective for treating HER2-overexpressing invasive breast cancer. In

Lovastatin enhances the antiproliferative effect of trastuzumab in HER2 over expressing breast cancer cells

2546 Background: Trastuzumab, a recombinant humanized anti-HER2 monoclonal antibody, has been found to have potent antiproliferative effects in HER2 overexpressing human breast tumors. Lovastatin, an HMG-CoA reductase inhibitor suppresses growth, and induces apoptosis in breast cancer cells. Both agents induce a G0/G1 arrest of cell cycle progression. The purpose of this study was to evaluate the antiproliferative effect of the novel combination of lovastatin and trastuzumab in human breast cancer cell lines. Methods: Increasing doses of lovastatin (0.16-40 μM) and trastuzumab (0.039-1 μg/ml) were tested alone and in combination. Three breast carcinoma cell lines were studied: two HER2 overexpressing lines (BT474 and SKBR3) and one cell line that expresses low levels of the receptor (MCF7). Inhibition of growth was assessed after 7 days of treatment by the MTT colorimetric assay, while apoptosis was detected using an in situ DNA Fragmentation Detection kit. Results: A dose-dependent growth inhibition was demonstrated in all three cell lines tested with lovastatin, while trastuzumab was only effective in the HER2 overexpressing cells. Trastuzumab (0.06 μg/ml) inhibited cell growth by 29.3% and 40.1%, while lovastatin (2.5 μM) caused an inhibition of 19.1% and 43% in the BT474 and SKBR3 cell lines, respectively. The combination of trastuzumab and lovastatin, at these doses, significantly inhibited growth in BT474 and SKBR3 cells by 45.4% and 68.9% (P < 0.001, oneway ANOVA) compared to either agent alone. Conclusions: Lovastatin plus trastuzumab treatment led to increased growth inhibition in HER2 positive breast cancer cell lines. Further studies in an animal model are warranted. This combination may also have important clinical therapeutic implications. No significant financial relationships to disclose.

SERUM LEVELS OF SHED HER2/NEU PROTEIN IN MEN WITH PROSTATE CANCER CORRELATE WITH DISEASE PROGRESSION

We determined the association between serum levels of shed Her-2/neu protein and disease progression in men with prostate cancer. Serum from 279 patients enrolled in a prospective serum bank and database at New York University Medical Center was analyzed using the Food and Drug Administration approved Immuno-1 Her-2/neu assay. Patients were classified by the Prostate-Specific Antigen Working Group model into 5 groups, namely group 1-no evidence of cancer in 60, group 2-clinically localized disease in 67, group 3-prostate specific antigen increasing after therapy and no clinical metastases in 77, group 4-clinical metastases and castration sensitivity in 42, and group 5-clinical metastases and castration resistance in 33. A cutoff of 14 ng/ml for normal serum Her-2/neu was established based on the 95th order statistic in group 1. Of 279 patients 37 (13.3%) had increased serum Her-2/neu, that is 5%, 11.9%, 10.4%, 16.7% and 33.3% in groups 1 to 5, respectively. There was a significant difference between patients with (groups 4 and 5) and without (groups 2 and 3) clinical metastases (p = 0.006). In group 5 patients serum Her-2/neu was significantly higher than in group 2 patients (p <0.02). The risk of cause specific death increased significantly with each unit increase in serum Her-2/neu (p <0.001). Increased serum Her-2/neu correlates with the presence of metastatic disease and it may indicate an increased risk of death in patients with castrate, metastatic prostate cancer. The detection of serum Her-2/neu is a minimally invasive alternative to tumor sampling for identifying potential candidates for anti-Her-2/neu treatment strategies. Further studies are needed to optimize this assay for application in the clinical setting.

Radiobromination of humanized anti-HER2 monoclonal antibody trastuzumab using N-succinimidyl 5-bromo-3-pyridinecarboxylate, a potential label for immunoPET

Combining the specificity of radioimmunoscintigraphy and the high sensitivity of PET in an in vivo detection technique could improve the quality of nuclear diagnostics. Positron-emitting nuclide 76Br ( T 1/2=16.2 h) might be a possible candidate for labeling monoclonal antibodies (mAbs) and their fragments, provided that the appropriate labeling chemistry has been established. For internalizing antibodies, such as the humanized anti-HER2 monoclonal antibody, trastuzumab, radiobromine label should be residualizing, i.e., ensuring that radiocatabolites are trapped intracellularly after the proteolytic degradation of antibody. This study evaluated the chemistry of indirect radiobromination of trastuzumab using N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate. Literature data indicated that the use of this method provided residualizing properties for iodine and astatine labels on some antibodies. An optimized “one-pot” procedure produced an overall labeling efficiency of 45.5±1.2% over 15 min. The bromine label was stable under physiological and denaturing conditions. The labeled trastuzumab retained its capacity to bind specifically to HER2-expressing SKOV-3 ovarian carcinoma cells in vitro (immunoreactivity more than 75%). However, in vitro cell test did not demonstrate that the radiobromination of trastuzumab using N-succinimidyl 5-bromo-3-pyridinecarboxylate improves cellular retention of radioactivity in comparison with the use of N-succinimidyl 4-bromobenzoate.

Targeting c-erbB2 and Other Receptors of the C-ErbB Family: Rationale and Clinical Applications

The c-erbB family of receptors includes four distinct receptors, namely c-erb B1, 2, 3 and 4 (HER1, 2, 3 and 4, respectively). Trastuzumab (T) is a recombinant humanized anti-HER2 monoclonal antibody that binds the extracellular domain of the receptor and blocks intracellular signalling. In clinical studies of T, either alone or in combination with chemotherapy, in HER2 overexpressing metastatic breast cancer patients, a significant benefit was obtained—improved response rates and survival, when T was combined with chemotherapy. Several trials of adjuvant T, either singly or in combination with chemotherapy, are in progress in early breast cancer patients. Pertuzumab defines a new class of HER2 inhibitors, “dimerization inhibitors” that block both homo- and hetero-dimerization of HER2. In preclinical studies pertuzumab is inhibitory to breast, prostate and non small cell lung cancer cell lines, both over and non overexpressing HER2. In phase I clinical trials pertuzumab has shown activity in a number of human cancers. A phase II program is in progress.

The HER receptor family: a rich target for therapeutic development

PurposeThe key role of the HER family of receptors in cancer has been widely acknowledged. HER receptor activation occurs via ligand binding or nonligand-dependent receptor dimerization, initiating signals that promote tumorigenesis via cell proliferation, survival, migration, adhesion, and differentiation. Therapeutic strategies designed to target and inhibit HER activation that are in clinical development are reviewed, including examples of both small-molecule tyrosine kinase inhibitors and monoclonal antibodies. Materials and methodsA literature review. ResultsTarceva is a potent, highly selective, reversible inhibitor of HER1/epidermal growth factor receptor tyrosine kinase with inhibitory activity against various in vitro and in vivo models of solid human tumors. Phase II trials in refractory non–small-cell lung, head-and-neck, and ovarian cancer have demonstrated clinical activity, including objective responses and prolonged, stable disease. Four Phase III trials are ongoing evaluating primarily the effect on survival of Tarceva in combination with chemotherapy. 2C4 is a humanized anti-HER2 monoclonal antibody that binds to a broad, extracellular epitope, resulting in steric inhibition of HER–receptor complex formation that involves HER2. 2C4 has shown significant activity in xenograft models of prostate, lung, and breast cancer. 2C4's activity, unlike Herceptin's, is not dependent on HER2 amplification. This antibody is in early clinical development. ConclusionThe strategy of targeting the HER system has been further validated by early experience with Tarceva and 2C4. The optimal clinical benefit of these agents will likely involve combinations of biologic agents, with or without traditional chemotherapy, and will be guided by critical predictive diagnostic information.

Efficacy and safety of trastuzumab as a single agent in heavily pretreated patients with HER-2/neu-overexpressing metastatic breast cancer.

The human epidermal growth factor receptor 2 (HER2) protein is a unique and useful target for antibody therapy against breast cancers that overexpress the HER-2/neu gene. The recombinant humanized anti-HER2 monoclonal antibody, trastuzumab, was approved for clinical use in the United States in 1998. It became available in Japan in June 2001. This study focuses on the efficacy and safety of trastuzumab as a single agent in second-third line treatment of HER2/neu-overexpressing metastatic breast cancer. Between June 2001 and May 2002, we treated 62 patients with trastuzumab, as a single agent or in combination chemotherapy, for second-third line treatment of HER2-overexpressing metastatic breast cancer. Twenty-seven of 62 patients were treated with trastuzumab as a single agent. We reviewed retrospectively the efficacy and safety of the drug given as a single agent. The expression of HER2 was determined by immunohistochemical staining. All patients received a standard loading dose of 4 mg/kg followed by 2 mg/kg weekly. Patients received a median of 16.7 weekly infusions (range, 1-66 infusions). Trastuzumab therapy was generally well tolerated. Clinically severe adverse events (grade 3 or 4) included hypotension (7.4%), and hypoxia (3.7%). Grade 1 to 2 toxicity included fever (11.1%) and diarrhea (3.7%). Infusion-related reactions were infrequent, as were serious hematologic complications. Cardiotoxicity did not occur in the study. Three patients had a complete and 3 a partial response, 3 had no change, 17 had progressive disease, and one was not evaluated. The overall response rate in the 26 patients with available data was 23.1% (95% confidence interval, 5.7-40.4). The median duration of response was 6.4 months (range, 2.5-14.0). The median time to progression was 3.1 months (range, 0.2-16.7). Response rates differed by metastatic site as follows: lung 0% (0/12), bone 10.0% (1/10), liver 0% (0/8), skin 50.0% (4/8), lymph nodes 42.9% (3/7), brain 0% (0/2). Molecular target therapy with trastuzumab appears safe and is generally well tolerated. For treatment of metastatic breast cancer, single agent therapy produces a durable response in some patients but lacks sufficient efficacy. Single agent use of trastuzumab is a viable option for treatment in cases with non-life-threatening disease without visceral metastasis.