HER2-expressing Cell Lines Research Articles

Role of CD24 in HER2-positive breast cancer.

e11572 Background: Human epidermal growth factor receptor 2 (HER2) is a transmembrane kinase receptor, which promotes the RAS/RAF/MEK/ERK and the PI3K/AKT/mTOR pathways. The standard treatment for HER2-positive breast cancer is a combination of chemotherapy plus an anti-HER2 agent, such as trastuzumab and lapatinib. HER2-positive breast cancers are predominantly positive for CD24, which is a glycosyl phosphatidylinositol (GPI)-anchored membrane protein. Overexpression of CD24 in breast cancer is associated with a poor prognosis. We hypothesize that the co-expression of HER2 with CD24 is associated with the therapeutic resistance of HER2-positive breast cancer cells. Methods: We established HER2-expressing cell lines by introducing a wild type HER2 gene into MDA-MB-231 triple-negative (ER-/PgR-/HER2-) breast cancer cells. We used the HER2-positive breast cancer cell lines BT-474, HCC202, SKBR3, and HCC1569. To investigate the relevance of CD24 expression in HER2-positive breast cancer, we analyzed HER2 and...

Abstract P6-05-08: Nuclear HER3 localisation plays a role in trastuzumab resistance in HER2-positive breast cancer

Abstract Background: HER3 is known to locate in the nucleus. Unlike HER4, nuclear HER3 translocation has not been reported to be due to a proteolytic cleavage process by ADAM17 and gamma-secretase. The mechanisms of nuclear HER3 induction and its role in relation to trastuzumab treatment and resistance for HER2-positive breast cancer is unclear. Methods: Using nuclear fractionation and confocal microscopy, nuclear HER3 localisation was investigated in response to trastuzumab with or without ADAM17 inhibitor and gamma-secretase inhibitor in a panel of HER2 expressing cell lines. We also correlated nuclear HER3 expression by immunohistochemistry with treatment response in patients who underwent window trastuzumab study as well as the survival outcome in a cohort of HER2-positive breast cancer patients using Kaplan–Meier survival curves with Log-rank test. Results: HER3 ligand heregulin and trastuzumab was found to induce nuclear HER3 translocation in HER2-positive breast cancer cell lines, including SKBR3. Nuclear HER3 was also enriched in acquired trastuzumab resistant SKBR3 cells (SKBr3-TR). Trastuzumab treatment induced several HER3 fragments and HER3100kD was found to be responsible for nuclear HER3 enrichment by fractionation. This fragment was confirmed to be a specific band of HER3 as shown by HER3 knockdown. Nuclear HER3 was reduced by inhibiting either gamma-secretase or ADAM17 inhibitor. Gamma-secretase or ADAM17 inhibitor reduced HER3100kD in both SKBr3 and SKBr3-TR cells. In HER2-positive breast cancer patients who underwent window trastuzumab study, baseline nuclear HER3 status was not a predictor of response for trastuzumab monotherapy at day 21. However, nuclear HER3 was enriched after trastuzumab treatment in a poor-responder patient. Total HER3 expression level in cytoplasm positively was correlated with poor response to trastuzumab monotherapy in HER2-positive patients (r = 0.67, p = 0.05). There was no statistically significant difference in disease-free survival between positive and negative nuclear HER3 expression but the number of patients was small (n = 87). Further validation to assess nuclear HER3 expression as a prognostic and predictive biomarker in HER2-positive breast cancer patients undergoing trastuzumab treatment will be assessed in randomized tumour samples from FinHER study. Conclusion: Heregulin and trastuzumab treatment seems to induce nuclear HER3 translocation in some of the HER2 positive breast cancer cells. This may be due to proteolytic cleavage of HER3 as it is reduced by ADAM17 or gamma-secretase inhibitor. Enriched nuclear localisation of HER3 seems to be one possible mechanism of acquired resistance to trastuzumab in HER2-positive breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-05-08.

Abstract B087: HER-2/neu expression regulates the immune response and tumor senescence in breast cancer

Abstract Background: Over-expression of the receptor tyrosine kinase ErbB2 (HER-2) has been widely implicated in malignant transformation, cell survival, motility and invasion in breast cancers. Intermediate expression of HER-2 has been identified in luminal breast cancer stem cells. HER-2 over-expression down regulates major histocompatibility complex class I molecules (MHCI) expression in breast cancer cells. Results: We investigated the expression of HER-2 in various HER-2 expressing cell lines and measured HLA A-2 MHC class I expression. High Her-2 expressing cell lines SKBR3 and SKOVA3 and intermediate expressing cell lines MCF7 demonstrated extremely low HLA A-2 expression, while low Her-2 expressing MDAMB231 demonstrated low HER-2 expression and high HLA A2 expression. Anti-HER-2 CD8 T cells recognize the latter cells but not high or intermediate expressing HER-2 expressing cells. Treatment of intermediate expressing cell lines but not high HER-2 expressing cells with interferon-gamma; (IFN- gamma) and TNF-alpha; result in markedly increased HLA A2 expression and 3 fold increase in CD8 T cell killing of the MCF7 cells. Treatment of high expressing HER-2 cells with trastuzumab but not lapatinib decreased HER-2 expression and combined with IFN-gamma; and TNF-alpha; result in increased HLA- A2 expression and dramatic increase in CD8 T cell recognition. In contrast, high and intermediate HER-2 expressing cells are sensitive to lysis and tumor senescence induction when treated with combinations of IFN-gamma; and TNF-alpha; in a dose dependent manner. Low HER-2 expressing cells are less sensitive to senescence induction. Conclusions: In conclusion, our results establish a potential role for HER-2 in regulating the immune response against breast cancer and suggest that CD8 T cells are more effective in eliminating low and intermediate HER-2 expressing cells while CD4 T cell derived IFN-gamma; and TNF-alpha; together with trastuzumab may be more effective in eliminating high expressing HER-2 breast cancer cells and inducing tumor senescence preventing recurrence. Citation Format: Cinthia Rosemblit, Jessica Cintolo-Gonzalez, Shuwen Xu, Brian J. Czerniecki. HER-2/neu expression regulates the immune response and tumor senescence in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B087.

Interaction between Her2 and Beclin-1 Proteins Underlies a New Mechanism of Reciprocal Regulation

Beclin-1 is a key regulator of autophagy that functions in the context of two phase-specific complexes in the initiation and maturation of autophagosomes. Its known interacting proteins include autophagy effectors, Bcl-2 family members, and organelle membrane anchor proteins. Here we report a newly identified interaction between Beclin-1 and the protein tyrosine kinase receptor Her2. We demonstrate that in Her2-expressing breast carcinoma cells that do not succumb to lapatinib, this Her1/2 inhibitor disrupts the cell surface interaction between Her2 and Beclin-1. The data suggest that the ensuing autophagic response is correlatively associated with the release of Beclin-1 from its complex with Her2 and with the subsequent increase in cytosolic Beclin-1. Upon its interaction with Her2, Beclin-1 up-regulates the phosphorylation levels of Her2 and Akt. The Beclin-1 evolutionarily conserved domain is required both for the interaction of Beclin-1 with Her2 and for the increased Her2 and Akt phosphorylation. These findings shed new light on mechanisms involved in lapatinib-mediated autophagy in Her2-expressing breast carcinoma cell lines and in Beclin-1 signaling in these cells.

Abstract 4331: Advantages of polyacetal polymer-based antibody drug conjugates employing cysteine bioconjugation.

Abstract The application of polymers to antibody drug conjugate (ADC) design and preparation can provide numerous advantages, including 1) significantly higher capacity for drug payload; 2) utilization of alternative payloads not suitable for direct conjugation approaches; 3) improvement of physicochemical properties of resulting ADCs; 4) utilization of protein recognition scaffolds beyond the commonly used IgGs; and 5) improvements in PK and biodistribution. Herein we present results of a novel, biodegradable polyacetal polymer-based conjugation system to create next-generation ADCs. The basis of this new conjugation system is a hydrophilic, fully biodegradable polyacetal carrier (PHF or poly(1-hydroxymethylethylene hydroxymethylformal) modified with chemically orthogonal linkers. One linker is used to covalently attach a targeting moiety (mAb or alternative) via cysteine conjugation, while a second, chemically distinct linker is used to attach a drug payload and to control the mechanism and rate of drug release. Previously we have reported highly efficacious polyacetal ADCs prepared by utilizing random lysine modification. In this report we present an alternative cysteine-based bioconjugation strategy. Conventional, direct drug-cysteine linked ADCs are destabilized due to loss of inter-chain disulfide bridges. In contrast, the polyacetal polymer conjugated via cysteines in the antibody hinge region stabilizes the resulting ADCs through the formation of inter-chain bridge structures. To demonstrate the benefits of this approach, we prepared Her-2 targeted ADCs with protein recognition scaffolds ranging in size from 15 kDa to 150 kDa, all targeting the Her-2 antigen, and bearing an anti-tubulin agent as the drug payload. ADCs were highly active and selective in vitro in Her-2 expressing cell lines. These polyacetal cysteine-based ADCs exhibited prolonged plasma and tumor exposure in the Her-2 expressing BT474 mouse xenograft model and tumor-specific accumulation. The ADCs were well-tolerated, and resulted in 100% tumor-free survivors. Citation Format: Alex Yurkovetskiy, Natalya Bodyak, Mao Yin, Joshua Thomas, Patrick Conlon, Cheri Stevenson, Alex Uttard, Liu Qin, Dmitry Gumerov, Elena Ter-Ovaneysan, Michael DeVit, Timothy B. Lowinger. Advantages of polyacetal polymer-based antibody drug conjugates employing cysteine bioconjugation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4331. doi:10.1158/1538-7445.AM2013-4331

Abstract 1271: A novel method to identify HER2-derived HLA-A2 epitopes for use in combination immunotherapy with Trastuzumab.

Abstract Introduction: Several MHC class I HER2-derived peptides have been identified that elicit a peptide-specific, cytotoxic T lymphocyte (CTL) response. We have postulated that Trastuzumab (Tz) binding may protect or modulate enzymatic cleavage sites on HER2 normally accessible by proteasomes thereby altering the cleavage pattern and resulting peptide profile. We have previously reported that Tz-treated breast cancer cells are recognized and killed more efficiently than untreated cells by T cells stimulated with the HER2-derived peptide E75 (aa:369-377). In the current study, we show that unique peptides may be located within the Tz binding sequence and that treatment with Tz may generate novel immunogenic peptides that could be used as vaccines in combination immunotherapy. Methods: The binding site of Tz on the HER2 protein was analyzed for HLA-A2 binding peptides. Five nonameric peptides were identified, synthesized and tested. HLA-stabilization assays using T2 cells were performed to confirm HLA-A2 binding. HER577 (aa:577-585; FGPEADQCV) demonstrated the highest affinity and was further studied. PBMC from healthy donors were stimulated with HER577 and tested in standard cytotoxicity assays (CTX) against HER2-expressing tumor cell lines that were untreated or pre-treated with Tz. NK cell depletion was performed to ensure that the effects were not due to ADCC. The effect of Tz treatment on cell surface MHC class I protein expression was also determined. Results: HER577 (aa:577-585; FGPEADQCV) showed binding affinity to HLA-A2 stronger than that of E75 (MFI = 532 for HER577 and 332 for E75). HER577 and E75 both had a n off-rate of t1/2 > 8 hr. CTX assays using MCF7 (HLA-A2+, low HER2) showed specific cytotoxicity by HER577-CTL (45±3%) that was similar to E75-CTL (51±4%). CTX assays evaluating Her577-CTL versus MCF7 cells pretreated with Tz resulted in a 16% increase in specific cytotoxicity. In MCF7 cells transfected to overexpress HER2 (HER18) the specific lysis was 30±5% which increased to 51±5% (p=0.05) with Tz treatment. Similarly, with SKBr3-A2 cells (SKBr3 cells transfected to express HLA-A2), the specific lysis was 44±5% which increased to 62±3% (p=0.02) with Tz treatment. Tz treatment of a panel of breast cancer cell lines with varying HER2 expression demonstrated no changes in HLA-A2 or HLA-ABC expression, confirming the increased killing after Tz treatment was caused by specific expression of the peptide/HLA-A2 complex. Conclusion: HER577 is a class I HER2-derived epitope identified using a novel method for the discovery of immunogenic peptides for vaccine development. Further investigation of combination immunotherapy with therapeutic antibodies and unique peptide vaccines is warranted. Citation Format: Na Qiao, Akhil Chawla, Gheath Alatrash, Anne Philips, George Peoples, Sathibalan Ponniah, Elizabeth A. Mittendorf. A novel method to identify HER2-derived HLA-A2 epitopes for use in combination immunotherapy with Trastuzumab. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1271. doi:10.1158/1538-7445.AM2013-1271

Postmenopausal breast cancer, androgens, and aromatase inhibitors

Recent data can help to better define the long debated relationship between androgens and breast cancer (BC) after menopause. We reviewed the available literature data on: the origin of androgens after menopause, the association between circulating androgens and BC incidence and recurrence, the relationship between circulating and intratumoral hormones, the prognostic significance of the presence of androgen receptors (ARs) in the different BC subtypes, the androgen effect on BC cell lines, and the relationship between androgens and aromatase inhibitors. Epidemiological, clinical, and preclinical data on the role of androgens and of ARs on estrogen receptor (ER)-negative BC are somewhat controversial. However, most preclinical studies suggest that activated ARs, when present, have a proliferative effect, particularly in HER2 expressing cell lines, due to the cross-talk between AR and HER2 pathways. As regards ER-positive BC, epidemiological studies associate androgen levels with increased incidence and risk of recurrences, whilst clinical studies associate the AR positivity with a better prognosis. Preclinical studies suggest that the action of androgens is bidirectional: mainly proliferative, because circulating androgens are the precursors of estrogens, but also anti-proliferative, because AR activation restrains ER activity. The relative increase of androgenic action that follows the blocking of androgen aromatization into estrogens by aromatase inhibitors (AIs), could contribute to their therapeutic efficacy in AR-positive cases. Available data, although defining a complex picture, suggest that circulating androgen levels are clinically relevant, particularly when AIs are used.

A gene expression profile indicative of early stage HER2 targeted therapy response

BackgroundEfficacious application of HER2-targetting agents requires the identification of novel predictive biomarkers. Lapatinib, afatinib and neratinib are tyrosine kinase inhibitors (TKIs) of HER2 and EGFR growth factor receptors. A panel of breast cancer cell lines was treated with these agents, trastuzumab, gefitinib and cytotoxic therapies and the expression pattern of a specific panel of genes using RT-PCR was investigated as a potential marker of early drug response to HER2-targeting therapies.ResultsTreatment of HER2 TKI-sensitive SKBR3 and BT474 cell lines with lapatinib, afatinib and neratinib induced an increase in the expression of RB1CC1, ERBB3, FOXO3a and NR3C1. The response directly correlated with the degree of sensitivity. This expression pattern switched from up-regulated to down-regulated in the HER2 expressing, HER2-TKI insensitive cell line MDAMB453. Expression of the CCND1 gene demonstrated an inversely proportional response to drug exposure. A similar expression pattern was observed following the treatment with both neratinib and afatinib. These patterns were retained following exposure to traztuzumab and lapatinib plus capecitabine. In contrast, gefitinib, dasatinib and epirubicin treatment resulted in a completely different expression pattern change.ConclusionsIn these HER2-expressing cell line models, lapatinib, neratinib, afatinib and trastuzumab treatment generated a characteristic and specific gene expression response, proportionate to the sensitivity of the cell lines to the HER2 inhibitor.Characterisation of the induced changes in expression levels of these genes may therefore give a valuable, very early predictor of the likely extent and specificity of tumour HER2 inhibitor response in patients, potentially guiding more specific use of these agents.

The HER2-Binding Affibody Molecule (ZHER2∶342)2 Increases Radiosensitivity in SKBR-3 Cells

We have previously shown that the HER2-specific affibody molecule (ZHER2∶342)2 inhibits proliferation of SKBR-3 cells. Here, we continue to investigate its biological effects in vitro by studying receptor dimerization and clonogenic survival following irradiation. We found that (ZHER2∶342)2 sensitizes the HER2-overexpressing cell line SKBR-3 to ionizing radiation. The survival after exposure to (ZHER2∶342)2 and 8 Gy (S8Gy 0.006) was decreased by a factor four compared to the untreated (S8Gy 0.023). The low HER2-expressing cell line MCF-7 was more radiosensitive than SKBR-3 but did not respond to (ZHER2∶342)2. Treatment by (ZHER2∶342)2 strongly increased the levels of dimerized and phosphorylated HER2 even after 5 minutes of stimulation. The monomeric ZHER2∶342 does not seem to be able to induce receptor phosphorylation and dimerization or sensitize cells to irradiation.

Abstract 3001: The expression of HER2 in human breast cancer cells leads to massive alteration of RNA polymerase II binding and gene activation

Abstract Background. Herceptin, which targets the protein HER2, has poor efficacy as a monotherapy. To better understand the role of HER2, we compared isogenic HER2 expressing and non-expressing cells for the distribution for RNA polymerase II (POL II) on promoters and RNA expression. Methods. Chromatin immunoprecipitation was performed with anti-RNA polymerase II (POL II) in high HER2-expressing and non-expressing MCF7 cells as well as two breast cancer cell lines with naturally amplified HER2; MDA453 and BT474. Binding of POL II was assessed on Agilent promoter arrays. Gene activation in the same cells was assessed by expression analysis using U133plus2 arrays. Results. De novo expression of HER2 in MCF7 cells causes POLII to interact strongly with 606 genes not bound in empty vector control cells (p < 0.05). 30% of these genes exhibited a significant increase in transcription (p < 0.05; Fold-change > 1.4). POL II also interacts 1079 genes that are common between HER2-expressing and control cells. Conversely, 678 genes were bound more tightly by POL II in the control (p < 0.05). 20% of these genes exhibited a significant decrease in transcription. An additional 825 genes, bind POLII “loosely” in MCF7HER2 cells but not in MCF7 cells. 30% of these genes were significantly differentially regulated relative to control. The transcription of 68 loosely bound genes was tested by qPCR for all three cell lines. When compared with the array data, the correlation coefficients were 0.74 - 0.90;(p < 0.01). In both HER2 and non-expressing cells, loosely bound POL II sites are largely associated with 3′ positions of the Agilent probes within the transcribed region, whereas strong binding sites were largely localized to + 250 bp of the transcription start sites (TSSs). The transcriptome of MCF7 was compared to MCF7HER2 and the two HER2-expressing cancer cell lines. 633 genes were differentially expressed between the control and the three lines. Conclusions. The expression of HER2 is associated with a marked alteration in the number of genes that interact with POL II, involving hundreds of genes. The resulting gene activity depends upon the binding strength of POL II. Strong localized binding in one part of the promoter is often associated with less transcription, and diffused looser binding is often associated with higher transcription, whereas a lack of POL II binding is usually associated with no transcription. This is consistent with a mass action model in which transcription is maximal for POL II sites with an optimal koff rate and declines with either an increase (stalled) or decrease (not interacting) in binding. 42%, of the HER2-dependent gene interactions and transcription changes occur in both of the human cancer cell lines with naturally amplified HER2. These observations reveal a much expanded role of HER2 in gene regulation and indicate a greatly extended role in gene activation in HER2-positive breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3001. doi:1538-7445.AM2012-3001

Mechanistic analysis of the antitumor efficacy of human natural killer cells against breast cancer cells

We investigated the role of human natural killer (NK) cells in the peripheral blood (PB) and liver in controlling breast cancer. The proportion of NK cells among liver mononuclear cells was significantly higher than among PB mononuclear cells. Liver NK cells inductively expressed higher levels of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) than PB NK cells in response to interleukin-2 (IL-2). Liver NK cells displayed higher cytotoxicity against various breast cancer cell lines (MDA-MB231, MDA-MB453, MDA-MB468, and MCF-7) after IL-2 stimulation than did PB NK cells. Anti-HER2 monoclonal antibody (mAb) promoted the cytotoxicity of both the types of NK cells toward HER2-expressing cell lines. All breast cancer cell lines highly expressed death-inducing TRAIL receptors, death receptor 4, but did not express death-inhibitory receptors (DcR1 and DcR2). Both PB and liver NK cell-induced cytotoxicity was inhibited partially by anti-TRAIL mAb and more profoundly by the combination of anti-TRAIL mAb and concanamycin A, indicating that TRAIL and perforin are involved. IL-2-stimulated liver and PB NK cells exhibited upregulated expression of CXCR3, which bind to the chemokines CXCL9, CXCL10, and CXCL11 secreted by breast cancer cells. We also found that IFN-γ promoted the production of CXCL10 from breast cancer cells. The results of this study show that IFN-γ secreted from NK cells likely promotes the production of CXCL10 from breast cancer cells, which in turn accelerates the migration of CXCR3-expressing NK cells into the tumor site. These findings suggest the possibility of a therapeutic approach by either activation of endogenous PB and liver NK cells or adoptive transfer of in vitro-activated autologous NK cells.

P1-12-08: Induction of Adhesion Molecules on Target Cells Maximizes Antibody-Dependent Cell-Mediated Cytotoxicity of Anti-Her2 Therapeutic Antibody with High Fucose Glycan.

Abstract Background: Antibody-dependent cell-mediated cytotoxicity (ADCC) is one pivotal effector function of certain therapeutic antibodies. ADCC is the lysis of antibody-coated target cells by effector cells expressing Fc receptors (FcR) that recognize the fragment crystallizable (Fc) region of the bound antibody. Higher fucosylated terminal glycan on Fc results in significantly lower ADCC activity because core fucose sterically hinders the binding of Fc region to FcR on effector cells. The role of adhesion molecules in ADCC is unclear but it has been reported that adhesion molecules help better orientation of effector cells (natural killer cells) toward adhesion molecule-coated beads. We hypothesize that a preferable association of effector cells and target cells mediated by adhesion molecules may compensate ADCC loss due to the steric hindrance of high core fucose on Fc. Recombinant humanized monoclonal anti-Her2 antibodies (anti-Her2 rhuMAb) at various fucosylation levels have been used in this study to evaluate the role of adhesion molecules in ADCC of antibodies with high fucose glycan. Materials and Methods: Natural killer (NK) cell line transfected with FcR were used as effector cells. Her2-expressing breast cancer cell lines BT-474 and SK-BR3 were used as target cells. Adhesion molecules were induced in target cells by incubation with 50ng/ml of recombinant human tumor necrosis factor (TNFa) for 1–2 days. Anti-Her2 rhuMAbs with different Fc fucosylation (G0-F from <1% to >2%) were added in the co-culture of effector cells and target cells. ADCC activities were quantitated by target cells lysis assessment using Europium-TDA release assay. Results: Untreated BT-474 or SK-BR3 target cells had very little adhesion molecule expression. A high fucosylated anti-Her2 rhuMAb induced 52% of target cell lysis, which represented two-thirds of cell lysis by a low fucosylated Ab. Increment of E:T ratio could not maximize ADCC activities. With TNFa treatment, target cell lysis was increased from 52% to 100% by high fucose rhuMAb. This enhancement of ADCC activities is probably due to the expression of adhesion molecules such as ICAM-1 and VCAM-1, which was shown to be up-regulated by 100-fold on target cells in the presence of TNFa. Our data suggested that ADCC activities in Her2-expressing breast cancer cell lines were HER2−specific, anti-HER2 rhuMAb-dependent and adhesion molecules were indispensible for the enhanced ADCC activities. Discussion: rhuMAbs produced in Chinese hamster ovary (CHO) cell lines may contain various Fc fucosylation, hence various ADCC activities. Although up-regulation of adhesion molecules alone cannot lyse target cells directly, our data suggested maximized ADCC activities by rhuMAbs with high Fc fucosylation level could be achieved by induction of adhesion molecule expression on target cells. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-12-08.

Effect of Microencapsulation Shear Stress on the Structural Integrity and Biological Activity of a Model Monoclonal Antibody, Trastuzumab

The aim of the present study was to investigate the influence of process shear stressors on the stability of a model monoclonal antibody, trastuzumab. Trastuzumab, at concentrations of 0.4–4.0 mg/mL, was subjected to sonication, freeze-thaw, lyophilisation, spray drying and was encapsulated into micro- and nanoparticles. The stressed samples were analysed for structural integrity by gel electrophoresis, SDS-PAGE, and size exclusion chromatography (SEC), while the conformational integrity was analysed by circular dichroism (CD). Biological activity of the stressed trastuzumab was investigated by measuring the inhibition of cell proliferation of HER-2 expressing cell lines. Results show that trastuzumab was resistant to the process shear stresses applied and to microencapsulation processes. At the lowest concentration of 0.4 mg/mL, a low percent (<9%) of soluble/reversible aggregates may have been formed. No loss of structural integrity, conformation was observed and no significant change in the biological activity of trastuzumab was observed (ANOVA; p > 0.05). The results of this study conclude that trastuzumab may be resistant to various processing stresses. These findings have important implications with respect to pharmaceutical processing of monoclonal antibodies.

Effective antibody therapy induces host-protective antitumor immunity that is augmented by TLR4 agonist treatment

Toll-like receptors are potent activators of the innate immune system and generate signals leading to the initiation of the adaptive immune response that can be utilized for therapeutic purposes. We tested the hypothesis that combined treatment with a Toll-like receptor agonist and an antitumor monoclonal antibody is effective and induces host-protective antitumor immunity. C57BL/6 human mutated HER2 (hmHER2) transgenic mice that constitutively express kinase-deficient human HER2 under control of the CMV promoter were established. These mice demonstrate immunological tolerance to D5-HER2, a syngeneic human HER2-expressing melanoma cell line. This human HER2-tolerant model offers the potential to serve as a preclinical model to test both antibody therapy and the immunization potential of human HER2-targeted therapeutics. Here, we show that E6020, a Toll-like receptor-4 (TLR4) agonist effectively boosted the antitumor efficacy of the monoclonal antibody trastuzumab in immunodeficient C57BL/6 SCID mice as well as in C57BL/6 hmHER2 transgenic mice. E6020 and trastuzumab co-treatment resulted in significantly greater inhibition of tumor growth than was observed with either agent individually. Furthermore, mice treated with the combination of trastuzumab and the TLR4 agonist were protected against rechallenge with human HER2-transfected tumor cells in hmHER2 transgenic mouse strains. These findings suggest that combined treatment with trastuzumab and a TLR4 agonist not only promotes direct antitumor effects but also induces a host-protective human HER2-directed adaptive immune response, indicative of a memory response. These data provide an immunological rationale for testing TLR4 agonists in combination with antibody therapy in patients with cancer.

Abstract 720: Mechanism of resistance to trastuzumab and molecular sensitization via ADCC activation by exogenous expression of HER2 extracellular domain in human breast cancer cells

Abstract Human epidermal growth factor receptor 2 (HER2) is a member of a family of receptors associated with tumor cell proliferation, apoptosis, adhesion, migration, and differentiation. According to numerous preclinical and clinical studies, HER2 can be immunogenic and antibodies against this transmembrane receptor have become in use in individuals with HER2-overexpressing tumors. Trastuzumab, a monoclonal antibody that targets HER2, induces antibody-dependent cellular cytotoxicity (ADCC) and inhibits HER2-mediated signaling. Numerous randomized trials have confirmed the utility of trastuzumab in HER2-overexpressing breast cancer in various clinical scenarios; however, HER2-overexpression has been identified only in approximately 25-30% of primary breast cancers. Moreover, it has been reported that the majority of metastatic breast cancer patients treated with trastuzumab develop acquired resistance within a year. In this study, we analyzed the mechanism of resistance to trastuzumab and examined for molecular sensitization by exogenous expression of HER2 extracellular domain in human breast cancer cells. A long-term exposure to trastuzumab induced its resistance in HER2-positive breast cancer cells. We found that trastuzumab-resistant cells exhibited down-regulation of HER2 expression in cell surface and impaired ADCC activity, suggesting that trastuzumab-resistant cells could be re-sensitized to trastuzumab by exogenous induction of extracellular domain (ECD) of HER2.We constructed the expression vector with HER2-ECD and stably transfected into low HER2-expressing human breast cancer cell line, MCF7. HER2-ECD expression had no apparent effect on the growth and signaling pathway in MCF7 cells; however, ADCC activity was strongly enhanced in HER2-ECD-expressing MCF7 cells compared to parental MCF7 cells. These results suggest that exogenous expression of HER2-ECD might be an appropriate strategy to sensitize HER2-negative or trastuzumab-resistant human breast cancer cells to trastuzumab. The modification of the tumor-specific replication-competent adenovirus (OBP-301, Telomelysin) to contain the HER2-ECD gene for exogenous expression is currently under the investigation. We will further examine the antitumor effects of this virus in the combination with trastuzumab. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 720. doi:10.1158/1538-7445.AM2011-720

Abstract 3616: Highly potent antibody-amanitin conjugates cause tumor-selective apoptosis

Abstract Alpha-amanitin blocks RNA polymerase II activity and is the most specific and most potent inhibitor of eukaryotic transcription. While widely used as a tool in molecular biology, only a few attempts have been made to employ alpha-amanitin as a therapeutic agent in oncology. The reason for this may be the high liver toxicity caused by the transport of amanitin into hepatocytes by the organic-anion transporter 1B3 (OATP1B3) present on hepatocytes, but not on other cell types. In this study we show that a direct impact on the transcription machinery can provide a novel approach in tumor therapy, provided that the amanitin can be targeted to tumor cells. This was achieved by using tumor-specific antibodies of high affinity armed with amanitin via suitable linker moieties. Using the HER2 specific monoclonal antibody Herceptin (Her) as a carrier, the antibody-drug conjugate (ADC) Her-DSC-30.0134 exhibits antiproliferative activity in HER2-expressing human tumor cell lines like SK-OV-3, SK-BR-3 and NCI-N87 in picomolar concentrations, indicating an up to 100.000-fold higher toxicity of the conjugate over non-conjugated alpha-amanitin. On cells lacking the HER2 receptor, e.g. HEK293 cells, toxicity of Her-DSC-30.0134 was not detected up to micromolar concentrations. This is also observed in HEK293 cells transfected with OATP1B3 (which incorporate amanitin similar as hepatocytes) indicating that amanitin-antibody-conjugates are no longer substrates for OATP1B3. These data suggest that a low hepatotoxicity of amanitin conjugates is expected. Subsequently, antitumoral activity of Her-DSC-30.0134 was evaluated in vivo in a s.c. xenograft mouse model using Herceptin-sensitive SK-OV-3 and Herceptin-resistant JIMT-1 cells. The ADC is well tolerated in nude mice and shows complete tumor remission in 7 out of 7 animals after a single i.v. injection in the SK-OV-3 and JIMT-1 models. By contrast, Herceptin shows only moderate antitumoral activity in the SK-OV-3 and no activity in the resistant JIMT-1 model, even after multiple injections. These data encourages further examination of amanitin-based antibody conjugates as highly potent and selective compounds for the treatment of various maligancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3616. doi:10.1158/1538-7445.AM2011-3616

Evaluation of a Maleimido Derivative of NOTA for Site-Specific Labeling of Affibody Molecules

Radionuclide molecular imaging has the potential to improve cancer treatment by selection of patients for targeted therapy. Affibody molecules are a class of small (7 kDa) high-affinity targeting proteins with appreciable potential as molecular imaging probes. The NOTA chelator forms stable complexes with a number of radionuclides suitable for SPECT or PET imaging. A maleimidoethylmonoamide NOTA (MMA-NOTA) has been prepared for site-specific labeling of Affibody molecules having a unique C-terminal cysteine. Coupling of the MMA-NOTA to the anti-HER2 Affibody molecule Z(HER2:2395) resulted in a conjugate with an affinity (dissociation constant) to HER2 of 72 pM. Labeling of [MMA-NOTA-Cys(61)]-Z(HER2:2395) with (111)In gave a yield of >95% after 20 min at 60 °C. In vitro cell tests demonstrated specific binding of [(111)In-MMA-NOTA-Cys(61)]-Z(HER2:2395) to HER2-expressing cell lines. In mice bearing prostate cancer DU-145 xenografts, the tumor uptake of [(111)In-MMA-NOTA-Cys(61)]-Z(HER2:2395) was 8.2 ± 0.9% IA/g and the tumor-to-blood ratio was 31 ± 1 (4 h postinjection). DU-145 xenografts were clearly visualized by a gamma camera. Direct in vivo comparison of [(111)In-MMA-NOTA-Cys(61)]-Z(HER2:2395) and [(111)In-MMA-DOTA-Cys(61)]-Z(HER2:2395) demonstrated that both conjugates provided equal radioactivity uptake in tumors, but the tumor-to-organ ratios were better for [(111)In-MMA-NOTA-Cys(61)]-Z(HER2:2395) due to more efficient clearance from normal tissues. In conclusion, coupling of MMA-NOTA to a cysteine-containing Affibody molecule resulted in a site-specifically labeled conjugate, which retains high affinity, can be efficiently labeled, and allows for high-contrast imaging.

Treatment of HER2-Expressing Breast Cancer and Ovarian Cancer Cells With Alpha Particle-Emitting 227Th-Trastuzumab

To evaluate the cytotoxic effects of low-dose-rate alpha particle-emitting radioimmunoconjugate (227)Th-p-isothiocyanato-benzyl-DOTA-trastuzumab ((227)Th-trastuzumab [where DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid]) internalized by breast and ovarian cancer cell lines in order to assess the potential of (227)Th-trastuzumab as a therapeutic agent against metastatic cancers that overexpress the HER2 oncogene. Clonogenic survival and cell growth rates of breast cancer cells treated with (227)Th-trastuzumab were compared with rates of cells treated with nonbinding (227)Th-rituximab, cold trastuzumab, and X-radiation. Cell growth experiments were also performed with ovarian cancer cells. Cell-associated radioactivity was measured at several time points, and the mean radiation dose to cells was calculated. SKBR-3 cells got 50% of the mean absorbed radiation dose from internalized activity and 50% from cell surface-bound activity, while BT-474 and SKOV-3 cells got 75% radiation dose from internalized activity and 25% from cell surface-bound activity. Incubation of breast cancer cells with 2.5 kBq/ml (227)Th-trastuzumab for 1 h at 4°C, followed by washing, resulted in mean absorbed radiation doses of 2 to 2.5 Gy. A dose-dependent inhibition of cell growth and an increase in apoptosis were induced in all cell lines. Clinically relevant activity concentrations of (227)Th-trastuzumab induced a specific cytotoxic effect in three HER2-expressing cell lines. The cytotoxic effect of (227)Th-trastuzumab was higher than that of single-dose X-radiation (relative biological effectiveness = 1.2). These results warrant further studies of treatment of breast cancer and ovarian cancer with (227)Th-trastuzumab.

A novel concept of adoptive immunotherapy with tumor necrosis factor-related, apoptosis-inducing, ligand-expressing natural killer cells for eliciting an anti-breast-cancer response.

e13048 Background: Previous studies have demonstrated that natural killer (NK) cells can destroy breast cancer cells. In the present study, we have reported that liver NK cells exhibit greater cytotoxic activity against tumor cells than do peripheral blood (PB) NK cells in humans. Methods: We investigated the phenotypic and functional properties of NK cells extracted from liver perfusates of living donors in clinical liver transplantation. Next, the susceptibility of various breast cancer cell lines to the activity of the liver and PB NK cells was tested. Results: Stimulation with IL-2 increased the expression of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on both the PB and liver NK cells. Accordingly, both the liver and PB NK cells exhibited strong cytotoxicity against various breast cancer cell lines (MDA-MB231, MDA-MB453, MDA-MB468, and MCF-7) after in vitro stimulation with IL-2. Further treatment with anti-HER2 monoclonal antibodies (mAbs) remarkably promoted the NK cell-mediated cytotoxicity toward the HER2-expressing breast cancer cell line MDA-MB453. All the breast cancer cell lines expressed remarkable levels of a death-inducing TRAIL receptor—death receptor (DR)4 and 5—which contains cytoplasmic death domains and mediates apoptosis, but they did not express the death-inhibitory receptors (DcRs) 1 and 2. Treatment with anti-TRAIL neutralizing mAbs partially reduced the liver and PB NK cell-mediated cytotoxicity toward breast cancer cells (32% ± 9%, 64% ± 15% reduction respectively at E:T = 5:1); this finding indicated the involvement of TRAIL-induced NK cell-mediated cytotoxicity. The expression of CXC chemokine receptor 3 (CXCR3), which binds to chemokines CXCL9, CXCL10, and CXCL11 secreted by breast cancer cells, was upregulated in the IL-stimulated NK cells. Conclusions: TRAIL+ NK cells kill breast cancer cells via death receptor/ligand interactions. This finding provides the basis for a novel concept, i.e., adoptive transfer of IL-2-stimulated NK cells to breast cancer patients for eliciting an anti-breast-cancer response. No significant financial relationships to disclose.

Effect of trastuzumab on antibody-dependent cellular cytotoxicity (ADCC) in HER2 nonamplified (non-amp) breast cancer (BC) cells.

659 Background: Trastuzumab (T) is currently indicated for the treatment of Her-2 positive breast cancers. The NSABP B-31 adjuvant trial suggested a possible clinical benefit for the addition of T to chemotherapy in patients whose tumors were Her-2 negative (low Her-2 expressing, non-amplified by FISH). ADCC mediated by CD56+ natural killer (NK) cells is known to play a role in response to T in Her-2 positive tumors. To investigate the possibility that T may induce ADCC against Her-2 negative tumors, we examined NK cell-mediated ADCC in T-treated, non-amp, low Her-2 expressing BC cell lines. Methods: A membrane/cytosol separation kit and immunoblotting were used to assess surface Her-2 levels in the non-amp BC cell lines CAMA-1, T47D, EFM19, MCF-7 and CAL-51. The positive control for Her-2 expression and amplification was HCC1954 and MDA-MB-468 was the negative control. PBMCs were isolated from whole blood of 8 healthy volunteers using a Ficoll-Plaque method. CD56+ NK cells were isolated from the PBMC fraction using magnetic bead based positive selection. Direct NK activity against the K562 cell line and the ability of the NK cells to induce T-related ADCC were measured using flow cytometry. Results: Membrane expression of Her-2 was detected in all cell lines examined except the MDA-MB-468 cell line. The direct cytotoxicity of NK cells ranged from 42% to 73% (ratio 10:1, NK: K562). T induced a significant ADCC response in the positive control HCC1954 (p value 0.002) and in each of the five non-amp, low Her-2 expressing cell lines, but not in the negative control MDA-MB-468 (Table). Conclusions: These results suggest that Her-2 non-amp BC cells, with low but detectable levels of Her-2 protein, can bind T and initiate ADCC. Future research will include determination of T-induced ADCC response by NK cells from patients with Her-2 non-amp BC. % increase in ADCC in response to 200 nM T (Ratio 1:1, NK: Target cell) HCC1954 CAMA-1 T47D EFM19 MCF-7 CAL-51 MDA-MB-468 Total Her-2 +++ + + + + + - % Increase ADCC ± std. dev. 29 ± 3 43 ± 13 21 ± 3 25 ± 7 31 ± 15 33 ± 8 0.6 ± 1 p value* 0.002 0.019 0.037 0.015 0.026 0.005 0.627 * p value – Student's t test, % ADCC for each cell line ± T in triplicate p<0.05 significant. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Roche Roche