anti-ErbB2 Antibody Research Articles

Effects of a human compact anti-ErbB2 antibody on gastric cancer

Gastric cancer represents one of the most common causes of cancer deaths worldwide. Overexpression of ErbB2, a tyrosine kinase receptor involved in the pathogenesis of several human cancer types, has been reported also in gastric cancer. Thus, the inhibition of ErbB2 signal transduction pathways by the use of human antibodies could be a valuable strategy for the therapy of this type of cancer. We tested for the first time the antitumor effects on gastric cancer cells of Erb-hcAb, a novel fully human compact antibody, prepared in our laboratory, which targets a different epitope of ErbB2 with respect to trastuzumab, the only anti-ErbB2 antibody currently in clinical use for both breast and gastric cancer therapy. Herein we demonstrate that the in vitro and in vivo growth of gastric cancer cells is efficiently inhibited by Erb-hcAb, which shows antitumor effects on the NCI-N87 cell line more potent than those observed for trastuzumab. Erb-hcAb could be a promising candidate in the immunotherapy of gastric cancer as it combines the antiproliferative effect associated with the inhibition of ErbB2 signaling on tumor target cells with the ability to induce antibody-dependent cellular cytotoxicity.

Identification of Caveolin-1 as a Potential Causative Factor in the Generation of Trastuzumab Resistance in Breast Cancer Cells

The oncogenic tyrosine kinase receptor ErbB2 is a prognostic factor and target for breast cancer therapeutics. In contrast with the other ErbB receptors, ErbB2 is hardly internalized by ligand induced mechanisms, indicating a prevalent surface expression. Elevated levels of ErbB2 in tumor cells are associated with its defective endocytosis and down regulation. Here we show that caveolin-1 expression in breast cancer derived SKBR-3 cells (SKBR-3/Cav-1) facilitates ligand induced ErbB2 endocytosis using an artificial peptide ligand EC-eGFP. Similarly, stimulation with humanized anti ErbB2 antibody Trastuzumab (Herceptin) was found to be internalized and co-localized with caveolin-1 in SKBR-3/Cav-1 cells. Internalized EC-eGFP and Trastuzumab in SKBR-3/Cav-1 cells were then delivered via caveolae to the caveolin-1 containing early endosomes. Consequently, attenuated Fc receptor mediated ADCC functions were observed when exposed to Trastuzumab and EC-Fc (EC-1 peptide conjugated to Fc part of human IgG). On the other hand, this caveolae dependent endocytic synergy was not observed in parental SKBR-3 cells. Therefore, caveolin-1 expression in breast cancer cells could be a predictive factor to estimate how cancer cells are likely to respond to Trastuzumab treatment.

Abstract P1-05-06: A novel mutation in the tyrosine kinase domain of ErbB2: molecular and proteomic investigation of its role in breast cancer invasion

Abstract Introduction: A landmark genomic study revealed 17 somatic coding mutations in a lobular metastatic breast tumor, none of which were present in the patient's primary tumor. Among these was a novel mutation in the tyrosine kinase domain of ErbB2, a gene often amplified in breast cancer. Other mutations in this domain have been shown to stimulate transformation, growth and invasion in breast cancer cells. We hypothesize that an accumulation of mutations in the primary tumor over time preceded the development of an invasive clone, which allowed it to metastasise. Therefore, we investigated the specific effects of ErbB2 on cellular signalling and invasion, in conjunction with proteomic analyses to show its effects on global protein expression. Methods: Wild-type and mutant ErbB2 cDNAs were cloned into V180 pLP 3X FLAG-tagged expression vectors. T47D breast cancer cells were transfected with one of these constructs. Protein was detected by immunoblotting with an anti-ErbB2 antibody. Membranes were then immunoblotted with an anti-phospho-ERK1/2 antibody. Transfected cells were seeded onto Matrigel; cells that migrated through the matrix were stained and counted after 48h. To identify global proteomic changes caused by mutant ErbB2, T47D cells transfected with mutant/wild-type ErbB2 or vector control, were differentially labeled by growing them in medium containing various lysine/arginine isotopes (known as stable isotope labeling of amino acids in cell culture; SILAC). Protein lysates were mixed and subjected to LC-MS/MS mass spectrometry. Proteomic changes were analysed using MaxQuant and DAVID software. Results: Overexpression of the wild-type or mutant ErbB2 genes produced protein expression in T47D, a non-invasive breast cancer cell line, confirmed by western blotting. To measure signalling downstream of ErbB2, we measured levels of the MAP kinase phospho-ERK1/2. Increased phosphorylation of ERK1/2 was demonstrated in cells expressing mutated ErbB2, compared with cells transfected with wild-type. This indicates increased activation of mutant ErbB2, demonstrating distinct phenotypic effects between wild-type and mutant ErbB2 in breast cancer cells. To analyze whether increased activation was accompanied by enhanced invasive properties, invasion assays were performed using Matrigel. A greater number of cells (1.5-fold increase) expressing mutant ErbB2 migrated across this matrix compared with wild-type-expressing cells. Global proteomic analysis of T47D cells overexpressing wild-type or mutant ErbB2 yielded 1,357 total proteins, of which 543 could be quantified. Two subsets of proteins were identified, whose levels were either increased or decreased, in the mutant versus wild-type-transfected lysates (compared to control ratios). Proteins elevated in the mutant-expressing cells included candidates involved in cell migration, inhibition of apoptosis and promotion of cellular survival. Proteins showing reduced expression were involved in pathways including adhesion and DNA damage response. Conclusion: This previously undescribed mutation in the tyrosine kinase domain of ErbB2 differentially effects downstream signalling pathways and may play a role in invasion of breast cancer cells. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-06.

Abstract P5-19-01: Targeting the HER3-phosphatidyl inositol-3 kinase pathway in breast cancers

Abstract Background: Using a mouse model of HER3 ablation in the mammary epithelium, we previously showed that HER3 is required for the genesis of transgenic HER2-overexpressing breast cancers. However, increasing evidence suggests that transgenic models of HER2 overexpression result in luminal B-like mammary tumors, which resemble human breast tumors that express estrogen receptor (ER) but are often not responsive to endocrine therapy. Furthermore, we found that HER3 is required in the untransformed mouse mammary epithelium for differentiation and survival of luminal cells, but not basal cells. Herein we report that HER3 drives growth and survival of luminal breast cancers in a manner that parallels its role in maintaining the untransformed luminal epithelium in the breast. Results: Expression profiling of a large human breast cancer dataset demonstrated that HER3 expression was highest in Luminal breast cancers as compared to different molecular subtypes of breast cancers, including HER2-enriched, claudin-low, and basal-like breast cancers. In luminal breast cancer cell lines MCF7, ZR75-1, and MDA-MB-361, HER3 targeting using siRNA or a monoclonal antibody against HER3 decreased Akt, S6, and Erk1/2 phosphorylation, cell growth and cell survival under conditions of estrogen deprivation. Luminal breast tumor xenografts treated with U3-1287, a humanized monoclonal anti-ErbB3 antibody, or EZN-3920, an anti-sense oligonucleotide against ErbB3, decreased tumor growth and phosphatidyl inositol-3 kinase (PI3K)/mTOR signaling in vivo. Interestingly, luminal breast tumor xenografts responded to the ER inhibitor fulvestrant with increased HER3 expression and phosphorylation, and with increased PI3K/mTOR signaling as shown by enhanced S6 phosphorylation. Fulvestrant-mediated upregulation of HER3 was also seen in matched clinical breast tumor specimens harvested before treatment and after 21 days of fulvestrant treatment. AMG-888 blocked fulvestrant-induced HER3 and phospho-S6 upregulation in vivo, resulting in regression of luminal breast tumor xenografts. Conclusions: These data suggest that HER3 targeting may improve the outcome of luminal breast cancers treated with endocrine therapy, and are consistent with our findings that a HER3-dependent expression signature positively correlates with relapse in tamoxifen-treated patients. Taken together, these results define a novel role for HER3 in luminal breast cancer cells, and support future investigations into therapeutic strategies to target HER3 in luminal breast cancers. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-19-01.

141 MM-121, an Anti-ErbB3 Antibody, Inhibits PI3K/AKT Signaling and Viability in Platinum-resistant Ovarian Cells and in Primary Ascites Derived From Chemo-resistant Ovarian Cancer Patients

141 MM-121, an Anti-ErbB3 Antibody, Inhibits PI3K/AKT Signaling and Viability in Platinum-resistant Ovarian Cells and in Primary Ascites Derived From Chemo-resistant Ovarian Cancer Patients

Gene Therapy of Malignant Solid Tumors by Targeting erbB2 Receptors and by Activating T Cells

One of the strategies to improve the outcome of anti-erbB2-mediated immunotherapy is to combine anti-erbB2 antibodies with T-cell-based adoptive immunotherapy, which can be achieved by expressing anti-erbB2 mAb on the surface of T cells. A single-chain variable fragment (scFv) from an anti-erbB2 mAb has been expressed on T cell surface to bind to erbB2-positive cells, and CD3ζ has been expressed as a fusion partner at C terminus of this scFv to transduce signals. T cells grafted with this chimeric scFv/CD3ζ were able to specifically attack target tumor cells with no MHC/Ag restriction. To test the effects of CD28 signal on cellular activation and antitumor effectiveness of chimeric scFv/CD3ζ-modified T cells, we constructed a recombinant anti-erbB2 scFv/Fc/CD28/CD3ζ gene in a retroviral vector. T cells expressing anti-erbB2 scFv/Fc/CD28/CD3ζ specifically lyzed erbB2-positive target tumor cells and secreted not only interferon-γ (IFN-γ) but also IL-2 after binding to their target cells. Our data indicate that CD3 and CD28 signaling can be delivered in one molecule, which is sufficient for complete T cell activation without exogenous B7/CD28 co-stimulation.

974PD - A Phase 1 Study of the Anti-Erbb3 Antibody Mm-121 in Combination with Weekly Paclitaxel in Patients with Advanced Gynecological and Breast Cancers

ABSTRACT Background MM-121 is a fully human monoclonal antibody targeting the epidermal growth factor receptor family member ErbB3. ErbB3 has been widely implicated in driving cancer growth and in the development of resistance to conventional chemotherapies and targeted agents across multiple malignancies. Single agent weekly paclitaxel is a standard regimen for patients with advanced gynecological and metastatic breast cancers. Here we present results from an ongoing Phase 1 study evaluating the combination of MM-121 and weekly paclitaxel. Methods A total of 24 patients with either platinum resistant ovarian cancer, metastatic endometrial cancer, or HER2 negative metastatic breast cancer, were treated in a dose escalation (12 pts) or expansion cohort (12 pts). Patients were treated until disease progression or intolerable toxicity. Response was assessed every eight (8) weeks according to RECIST 1.1. In the dose-expansion cohorts, pre- and post-treatment fresh tumor biopsies were obtained from 12 patients to assess ErbB3 signaling status and its potential as a predictive marker for MM-121 response. Results To date, 24 patients have been treated with a median follow up of 5.5 months (range 0.8 – 13.1). The median age was 58 years (range 38 – 72), and patients had received a median of four (range 1 – 11) prior lines of therapy. Common (>20%) adverse events of any grade included fatigue (62%), peripheral neuropathy (58%), diarrhea (46%), neutropenia (46%) and rash (38%). Grade 3/4 toxicities included fatigue (17%), peripheral neuropathy (8%), diarrhea (12%), neutropenia (16%), anemia (4%), abdominal pain (8%), and hypokalemia (4%). 17 (71%) patients were evaluable for response and the overall clinical benefit rate, defined as PR or SD lasting for >4 months, was 71%. 47% achieved a PR and 35% a confirmed PR with a median duration of response of 4.6 months (range1.7 – 9.6) and 24% had SD >4 months with a median duration of SD of 5.6 months (range 4.2-12.6). 17% patients had PD at first assessment and 38% remain on study with a median on-study time of 10.2 months (range 1.4 – 13.1). Translational analyses exploiting tissue analysis in combination with in silico modeling are ongoing. Conclusion The combination of MM121 and paclitaxel showed activity in metastatic ovarian and breast cancers. Disclosure J. Liu: Joyce Liu is an investigator participating in the corporate-sponsored study which is the subject of the abstract. R. Patel: Ravi Patel is an investigator participating in the corporate-sponsored study which is the subject of the abstract. G. Kato: Giraldo Kato is an investigator participating in the corporate-sponsored study which is the subject of the abstract. U. Matulonis: Ursula Matulonis is an investigator participating in the corporate-sponsored study which is the subject of the abstract. V. Moyo: Victor Moyo is employed by and owns stock in the corporate sponsor of this reserach. K. Riahi: Kaveh Riahi is employed by and owns stock in the corporate sponsor of this research. J. Pearlberg: Joseph Pearlberg is employed by and owns stock in a corporate sponsor of this research. A. Czibere: Akos Czibere is employed by and owns stock in the corporate sponsor of this research. S. Isakoff: Steven Isakoff is an investigator participating in the corporate-sponsored study which is the subject of the abstract. He has also served as a consulant to Merrimack Pharmaceuticals.

The promise of anti-ErbB3 monoclonals as new cancer therapeutics

In the last 3-5 years strong evidence has been gathered demonstrating ErbB3 as a key node for the progression of several cancer types. From the mechanistic standpoint the intracellular region of this receptor is rich of tyrosine residues that, upon phosphorylation, become high affinity binding sites for PI3K and other proteins involved in signal transduction. The involvement of ErbB3 occurs at different levels, most likely as a consequence of its promiscuity in the interaction with other RTKs of the same or other families. Several efforts are therefore being put in the development of antibodies that target this receptor either singly or in combination with other synergizing receptors. Some of these compounds have already entered clinical development. Although clinical proof-of-concept has not yet been achieved, this is likely to occur soon and will further accelerate the inclusion of anti-ErbB3 monoclonals in the repertoire of anticancer agents for more effective combination therapy. In this paper we review the wealth of anti-ErbB3 antibodies under development and compare their properties and potential to become marketed drugs.

Effective suppression of breast tumor growth by an anti-EGFR/ErbB2 bispecific antibody

Despite the effectiveness of the anti-ErbB2 humanized antibody trastuzumab, less than 35% of patients with ErbB2-overexpressing breast cancer respond to the treatment. Here we engineered an anti-EGFR/ErbB2 bispecific antibody (TC-BsAb) using trastuzumab and cetuximab, an anti-EGFR chimeric antibody. TC-BsAb treatment led to internalization of both EGFR and ErbB2, whereas trastuzumab and cetuximab, either alone or in combination, failed to induce ErbB2 internalization. Both in vitro and in vivo experiments indicated that TC-BsAb was significantly more potent in inhibiting the growth of breast cancer cell lines than trastuzumab, cetuximab, and trastuzumab plus cetuximab, suggesting its potential use for treating breast cancer.

Truncated p110 ERBB2 induces mammary epithelial cell migration, invasion and orthotopic xenograft formation, and is associated with loss of phosphorylated STAT5

Truncated-ERBB2 isoforms (t-ERBB2s), resulting from receptor proteolysis or alternative translation of the ERBB2 mRNA, exist in a subset of human breast tumors. t-ERBB2s lack the receptor extracellular domain targeted by therapeutic anti-ERBB2 antibodies and antibody–drug conjugates, including trastuzumab, trastuzumab-DM1 and pertuzumab. In clinical studies, expression of t-ERBB2 in breast tumors correlates with metastasis as well as trastuzumab resistance. By using a novel immuno-microarray method, we detect a significant t-ERBB2 fraction in 18 of 31 (58%) of immunohistochemistry (IHC)3+ ERBB2+ human tumor specimens, and further show that t-ERBB2 isoforms are phosphorylated in a subset of IHC3+ samples (10 of 31, 32%). We investigated t-ERBB2 biological activity via engineered expression of full-length and truncated ERBB2 isoforms in human mammary epithelial cells (HMECs), including HMEC and MCF10A cells. Expression of p110 t-ERBB2, but not p95m (m=membrane, also 648CTF) or intracellular ERBB2s, significantly enhanced cell migration and invasion in multiple cell types. In addition, only expression of the p110 isoform led to human breast epithelial cell (HMLE) xenograft formation in vivo. Expression of t-ERBB2s did not result in hyperactivation of the phosphoinositide kinase-3/AKT or mitogen-activated protein kinase signaling pathways in these cells; rather, phosphoproteomic array profiling revealed attenuation of phosphorylated signal transducer and activator of transcription 5 (STAT5) in p110-t-ERBB2-expressing cells compared to controls. Short hairpin-mediated silencing of STAT5 phenocopied p110-t-ERBB2-driven cell migration and invasion, while expression of constitutively active STAT5 reversed these effects. Thus, we provide novel evidence that (1) expression of p110 t-ERBB2 is sufficient for full transformation of HMEC, yielding in vivo xenograft formation, and (2) truncated p110 t-ERBB2 expression is associated with decreased phosphorylation of STAT5.

ErbB3 Inhibitory Surrobodies Inhibit Tumor Cell Proliferation In Vitro and In Vivo

ErbB3 is an important regulator of tumorigenesis and is implicated in development of resistance to several currently used oncology drugs. We have identified ErbB3 inhibitors based on a novel biologic scaffold termed a surrobody. Two of these inhibitors appear to work by a previously unrecognized mechanism of action. As a consequence, they not only inhibited cell proliferation and intracellular signaling driven by stimulation with the ErbB3 ligand neuregulin (NRG), but also inhibited signaling and proliferation that was driven by overexpression of ErbB2 in the absence of ligand stimulation. In addition, the surrobodies inhibited tumor growth in vivo in both ErbB2-overexpressing and nonoverexpressing cells. In ErbB2-overexpressing cells, both of the anti-ErbB3 surrobodies significantly augmented the activities of trastuzumab, lapatinib, and GDC-0941, agents that inhibit cell proliferation by different mechanisms. Moreover, although NRG diminished the efficacy of these agents, when they were combined with anti-ErbB3 surrobodies the affect of NRG was abrogated. In this capacity, the anti-ErbB3 surrobodies were more effective than the ErbB2/ErbB3 dimerization inhibitory antibody pertuzumab. Despite the fact that these surrobodies appear to engage ErbB3 differently than previously described anti-ErbB3 antibodies, they retain all of the beneficial characteristics of this class of agents, including the ability to augment drugs that inhibit EGF receptor. These anti-ErbB3 agents, therefore, show substantial promise for development as single agents or in combination with other ErbB-directed antibodies or small molecules and may provide for a broader range of therapeutic indications than previously described anti-ErbB3 antibodies.

Pertuzumab Increases 17-AAG-Induced Degradation of ErbB2, and This Effect Is Further Increased by Combining Pertuzumab with Trastuzumab

ErbB2 is an important oncogenic protein involved in carcinogenesis of, among others, breast, gastric, and ovarian carcinoma. Over-expression of ErbB2 is found in almost 20% of breast cancers, and this results in proliferative and anti-apoptotic signalling. ErbB2 is therefore an important treatment target. Antibodies recognizing full-length ErbB2 are clinically established, and drugs targeting the ErbB2 stabilizing heat shock protein 90 (Hsp90) are under clinical evaluation. We have investigated effects of the ErbB2-binding antibodies trastuzumab and pertuzumab alone and in combination, as well as the effect of the antibodies in combination with the Hsp90 inhibitor 17-AAG. Our results confirm the notion that combination of different ErbB2-binding antibodies more efficiently down-regulates ErbB2 than does one antibody in isolation. Additionally, our data demonstrate that ErbB2 is most efficiently down-regulated upon incubation with anti-ErbB2 antibodies in combination with Hsp90 inhibitors. The combination of anti-ErbB2 antibodies, and especially the combination of antibodies with 17-AAG, did also increase the inhibition of Akt activation of either agent, which could suggest an anti-proliferative effect. In such case, combining these agents could be beneficial in treatment of tumors not responding to trastuzumab only.

Novel anti‐ErbB3 monoclonal antibodies show therapeutic efficacy in xenografted and spontaneous mouse tumors

The role of the ErbB3 receptor in signal transduction is to augment the signaling repertoire of active heterodimeric ErbB receptor complexes through activating the PI3K/AKT pathway, which in turn promotes survival and proliferation. ErbB3 has recently been proposed to be involved in acquired resistance to tyrosine kinase inhibitors (TKIs), and is therefore a promising new drug cancer target. Since ErbB3 is a kinase defective receptor, it cannot be targeted by small molecule inhibitors, whereas monoclonal antibodies may offer a viable strategy for pharmacological intervention. In this study, we have utilized DNA electroporation (DNA-EP) to generate a set of novel hybridomas directed against human ErbB3, which have been characterized for their biochemical and functional properties and selected for their ability to negatively regulate the ErbB3-mediated signaling pathway. In vitro, the anti-ErbB3 antibodies modulate the growth rate of cancer cells of different origins. In vivo they show antitumoral properties in a xenograft model of human pancreatic tumor and in the ErbB2-driven carcinogenesis genetically engineered mouse model (GEMM) for mammary tumor, the BALB/neuT. Our data confirm that downregulating the ErbB3-mediated signals with the use of anti-ErbB3 monoclonal antibodies is both feasible and relevant for therapeutic purposes and provides new opportunities for novel anti-ErbB3 combinatory strategies for cancer treatment.

Mechanisms of cardiotoxicity associated with ErbB2 inhibitors

The ErbB2 receptor is a proto-oncogene associated with a poor prognosis in breast cancer. Herceptin, the only humanized anti-ErbB2 antibody currently in clinical use, has proven to be an essential tool in the immunotherapy of breast carcinoma, but induces cardiotoxicity. ErbB2 is involved in the growth and survival pathway of adult cardiomyocytes; however, its levels in the adult heart are much lower than those found in breast cancer cells, the intended targets of anti-ErbB2 antibodies. Furthermore, clinical trials have shown relatively low cardiotoxicity for Lapatinib, a dual kinase inhibitor of EGFR and ErbB2, and Pertuzumab, a new anti-ErbB2 monoclonal antibody currently in clinical trials, which recognizes an epitope distant from that of Herceptin. A novel human antitumor compact anti-ErbB2 antibody, Erb-hcAb, selectively cytotoxic for ErbB2-positive cancer cells in vitro and vivo, recognizes an epitope different from that of Herceptin, and does not show cardiotoxic effects both in vitro on rat and human cardiomyocytes and in vivo on a mouse model. We investigated the molecular basis of the different cardiotoxic effects among the ErbB2 inhibitors by testing their effects on the formation of the Neuregulin 1β (NRG-1)/ErbB2/ErbB4 complex and on the activation of its downstream signaling. We report herein that Erb-hcAb at difference with Herceptin, 2C4 (Pertuzumab) and Lapatinib, does not affect the ErbB2-ErbB4 signaling pathway activated by NRG-1 in cardiac cells. These findings may have important implications for the mechanism and treatment of anti-ErbB2-induced cardiotoxicity.

Therapeutic Use of an Anti-ErbB3 Monoclonal Antibody

Emerging evidence suggests that the catalytically inactive ErbB3 (HER3) protein plays a fundamental role in normal tyrosine kinase receptor signaling as well as in aberrant functioning of these signaling pathways, resulting in several forms of human cancers and some mental disorders. Here we report the generation of a specific anti-ErbB3 antibody intended for use in diagnosing disease or therapeutic application. By using the hybridoma technique, one cell line (2E(12)C(3)) stably producing anti-ErbB3 antibody was obtained. Its molecular weight was about 185 kDa and its isotype was IgG 2a and κ, respectively. The affinity constant (Kaff) of the anti-ErbB3 MAb was 5.83×10(10) M(-1). This antibody may become a useful tool for diagnostic and therapeutic targeting of ErbB3-expressing cancers or helpful in highlighting the etiology of schizophrenia.

Concurrent biological targeting therapy of squamous cell carcinoma of the esophagus with cetuximab and trastuzumab

The ERBB proteins are cell membrane tyrosine kinase receptors. Among these receptors, ERBB1 (EGFR or HER1) and ERBB2 (HER2/Neu) have been reported to be the most important in terms of the development and progression of squamous cell carcinoma of the esophagus (SCC). Thus, targeting of ERBB1 and ERBB2 may become a promising strategy to treat SCC. In the present study, we examined ERBB1 and ERBB2 expression of SCC cell lines (TT, TE2, TE6 and TE10) and tumor samples. In addition, we evaluated the effect of the anti-ERBB1 antibody cetuximab and the anti-ERBB2 antibody trastuzumab for SCC in vitro and in vivo. Biological activities (receptor downregulation, the phosphorylation of MAPK and AKT) induced by the two agents were also investigated. Immunohistochemistry of SCC samples showed that ERBB1 was detected in 84%, while ERBB2 was detected in 30%. RT-PCR analysis revealed that ERBB1 and ERBB2 mRNA were detectable in all four cell lines. MTT cell proliferation analysis showed that cetuximab, but not trastuzumab, inhibited growth in each of the SCC cell lines in a dose-dependent manner. Further, cetuximab and trastuzumab used together produced stronger inhibition of growth compared to cetuximab alone. Cetuximab downregulated ERBB1, but not ERBB2, at the TE6 cell surface. However, neither ERBB1 nor ERBB2 showed any downregulation by trastuzumab at the TE6 cell surface. Cetuximab, not but trastuzumab, inhibited the phosphorylation of MAPK and Akt. When administered in combination, the two agents inhibited Akt phosphorylation to a greater degree compared to treatment with cetuximab alone. In the in vivo study, cetuximab, but not trastuzumab, significantly inhibited the TT tumors. Additionally, the combination of cetuximab with trastuzumab induced a synergistic inhibitory antitumor effect in the TT tumors. In conclusion, combination of cetuximab and trastuzumab revealed a synergistic antitumor effect for SCC in vitro and in vivo. The antitumor effect may be induced by the inhibition of the phosphorylation of Akt. These findings suggest that combination therapy including cetuximab and trastuzumab may be a promising strategy to treat SCC.

Human recombinant lactoferrin as a new adjuvant in specific DNA vaccination against ErbB2 (75.2)

Abstract Lactoferrin triggers several innate immunity mechanisms. Repeated oral administrations of a recombinant human lactoferrin (Talactoferrin, TLF, Agennix, Houston, TX) hamper the growth of both transplantable tumors and autochthonous mammary carcinomas arising in BALB/c mice transgenic for the rat ErbB2 oncogene (BALB-neuT mice). To evaluate the mechanisms that delay the tumor growth and enhance the immune response elicited by a DNA vaccine against rat ErbB2 oncogene, BALB-neuT mice received by gavage PBS or TLF (1 g/kg) once daily, 5 days per week, 3 weeks on and 1 week rest for seven cycles. Following oral TLF we observed: decrease of serum IL4 and IL5; increase of serum IL12 and IFNγ; decrease of immature myeloid cells in tumor draining lymph nodes. When BALB-neuT mice receiving oral TLF were vaccinated with 50 µg of DNA plasmid coding for the extracellular and transmembrane domain of rat ErbB2 we found: extended tumor-free survivals; increased overall survivals; decrease in the number of tumors/mouse; higher titers of vaccine-induced anti-ErbB2 antibodies; similar cytotoxicity activities against ErbB2+ tumors. These findings suggest that TLF is an interesting new adjuvant in DNA vaccination against ErbB2.

Targeted therapies in breast cancer

Targeted therapies have improved cure rates and prolonged survival in metastasised breast cancer. The most important new molecular targets in breast cancer therapy are epidermal growth factor receptor (ErbB) family signalling, DNA repair pathways and angiogenesis. Blocking ErbB2 signalling with anti-ErbB2 antibodies or ErbB2 kinase inhibitors is effective in both the adjuvant and the palliative treatment of ErbB2 positive breast cancer. Poly-ADP-ribose polymerase (PARP) inhibitors lead to synthetic lethality in double strand repair deficient tumours. Anti-VEGF antibodies reduce tumour-induced angiogenesis and prolong progression-free survival in breast cancer. The use of both PARP inhibitors and antiangiogenic therapy is currently hampered by a lack of predictive biomarkers. In contrast, predictive markers are available for ErbB family signalling. This review is intended to give a concise summary of recent developments in the therapy of breast cancer with a focus on new, targeted therapies.

Abstract 2718: REGN1400, a fully-human ERBB3 antibody, potently inhibits tumor growth in preclinical models, both as a monotherapy and in combination with EGFR or HER2 blockers

Abstract ERBB3 is a member of the ERBB family of receptor tyrosine kinases, that includes EGFR and HER2, well-established drivers of tumorigenesis. Recent preclinical studies indicate that both EGFR and HER2 cooperate with ERBB3 to activate the phosphatidylinositol-3 kinase/Akt survival pathway and to promote tumor cell survival. Furthermore, ERBB3 activation has been demonstrated to mediate resistance to EGFR inhibitors in non-small cell lung cancer cell lines and to HER2 inhibitors in breast cancer cell lines. Consistent with these findings, anti-ERBB3 monoclonal antibodies have been shown to inhibit the growth of human tumor xenografts, demonstrating the importance of ERBB3 for tumor growth in vivo. REGN1400 is a fully-human anti-ERBB3 monoclonal antibody that binds human ERBB3 with high affinity (KD ∼50pM) and potently inhibits binding of the ERBB3 ligand neuregulin 1 (IC50 ∼30pM). REGN1400 inhibited phosphorylation of ERBB3 and Akt in multiple human tumor cell lines in vitro, including A431 (epidermoid carcinoma), MDA-MB-175-VII (breast cancer) and FaDu (head and neck cancer). In addition, REGN1400 strongly inhibited the growth of these cell lines in vitro. Consistent with its potent effects on tumor cell growth in vitro, REGN1400 strongly inhibited the growth of A431 and FaDu tumor xenografts in a dose-dependent manner, providing 70% and 97% tumor growth inhibition, respectively. Inhibition of FaDu tumor growth by REGN1400 was accompanied by a significant decrease in tumor ERBB3 phosphorylation, as assessed by both western blot and immunohistochemistry, confirming target inhibition in vivo. While treatment of FaDu tumors with single agent REGN1400 provided almost complete tumor growth inhibition, but not regression, the combination of REGN1400 plus anti-EGFR antibody caused significant tumor regression. Furthermore, combination treatment with REGN1400 plus the anti-HER2 antibody trastuzumab inhibited the growth of BT474 breast tumor xenografts more potently than either single agent. These findings indicate that ERBB3-directed therapies might be more effective when combined with agents that inhibit other ERBB family members. Finally, gene profiling studies in REGN1400-treated tumor cells revealed upregulation of several genes that could promote resistance to REGN1400, e.g., ERBB3 itself and ERBB4. Further analysis of tumor gene expression changes induced by REGN1400 could provide insight into potentially useful combination regimens. In summary, REGN1400, a novel anti-ERBB3 antibody, exhibits significant antitumor activity in preclinical models, suggesting the possibility that it could provide benefit to patients with multiple types of cancer, either as a single agent or when combined with therapies targeting other ERBB family members. 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 2718. doi:1538-7445.AM2012-2718

Abstract 4945: Targeting ErbB3-addicted cancers across the HER2 spectrum

Abstract ErbB3 is a critical activator of phosphoinositide 3-kinase (PI3K) signaling in EGFR-, HER2-, and MET-addicted cancers, and ligand-driven activation of ErbB3 is a prominent method by which cancers become resistant to targeted inhibitors of ErbB signaling, as well as anti-hormonal and chemotherapeutic agents. With the goals of inhibiting survival signaling in cancer cells, delaying the onset of resistance to standard-of-care therapies, and re-sensitizing cancer cells to prior therapies, we developed an anti-ErbB3 monoclonal antibody, MM-121, that effectively blocks ligand-dependent activation of ErbB3 and induces internalization and degradation of the receptor. The activity of single-agent MM-121 was examined in xenograft models and a machine-learning algorithm was developed that accurately predicts which preclinical models of cancer will respond to MM-121 based on five protein-based biomarkers. By simulating ErbB3 inhibition using a computational model of ErbB signaling, we predicted that a bispecific molecule that docks onto HER2 and subsequently binds to ErbB3 would effectively block ErbB3-driven signaling in HER2-amplified tumors. We therefore developed a bispecific antibody comprising two human scFv antibodies linked by a modified form of human serum albumin. The resulting molecule, MM-111, forms a trimeric complex with HER2 and ErbB3, effectively inhibiting ErbB3 signaling and demonstrating antitumor activity in preclinical models that are dependent on HER2 overexpression. Both MM-121 and MM-111 are now in early stages of clinical development. 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 4945. doi:1538-7445.AM2012-4945