Monoclonal HER2 Research Articles

Intrinsic HER2 V777L mutation mediates resistance to trastuzumab in a breast cancer patient

HER2 (ERBB2) is an oncogene and 20% of breast cancers display HER2 amplification. The HER2 monoclonal antibody, trastuzumab, is used to treat breast cancers that display HER2 amplification, with good responses in 80-90% of cases; however, 10% of tumours develop resistance to trastuzumab. In this study, we collected data of primary breast cancer patients who treated at hospital during 2004-2014. In our cohort, 205 of 1497 primary breast cancer patients showed HER2-amplification, and 20 experienced recurrence after trastuzumab therapy. Of the 20 recurrent cases, only six patients had metastatic sites, excluding brain metastases, which were resistant to trastuzumab. To examine trastuzumab resistance in HER2-amplified breast cancer, we analysed clinical specimens before and after trastuzumab therapy. The results indicated that an intrinsic activating mutation leads to a valine-to-leucine substitution at codon 777 within the HER2 kinase domain (HER2 V777L). This was identified in one of six cases of a HER2-amplified breast cancer, both pre- and post-treatment; however, HER2 V777L was not identified in 14 responders who were treated with trastuzumab. These results suggest that HER2 V777L mutation is responsible for, and a predictive marker of, trastuzumab resistance. This is the first report to show that HER2 V777L is coincident with HER2-amplification in breast cancers that have developed trastuzumab resistance.

Anthracycline- and trastuzumab-induced cardiotoxicity: a retrospective study.

Some chemotherapeutic agents cause cardiotoxic effects including reduction in left ventricular ejection fraction (LVEF) and occasionally congestive heart failure. Anthracyclines and HER2 monoclonal antibodies are common offenders, but clinical practice data on LVEF changes, risk factors and acute recovery is lacking. We retrospectively examined the electronic medical record at an academic medical center for receipt of anthracyclines and/or trastuzumab from 2000 to 2013 in cancer patients. Patient characteristics and serial LVEF assessments were collected. Patients with and without LVEF decline were analyzed by univariate and multivariate analysis. A total of 549 patients were identified with anthracycline/trastuzumab use and 216 had multiple LVEF assessments. Only 27 of the 216 patients who had multiple LVEF assessments at multiple occasions suffered a clinically significant LVEF fall (12.5%), and symptomatic CHF was rare (0.5%). Compared to unaffected patients, those with a fall in LVEF were more likely to have hypertension, hyperlipidemia or coronary artery disease (CAD). Concomitant trastuzumab and anthracycline use was a risk factor (36 vs 9.5% for anthracycline alone, p<0.001). The median time from start of chemotherapy to reduced LVEF was 202days (5-3008). On multivariate analysis, hypertension and use of trastuzumab remained independent predictors of LVEF fall. Acute recovery in LVEF was observed in 44% of patients. LVEF changes from cancer therapies are frequent and hard to predict. Hypertension, hyperlipidemia and CAD are associated with LVEF decline. Acute recovery of LVEF is observed in those experiencing treatment-related cardiotoxicity. Attention to timely interruption of cardiotoxic chemo is recommended.

A Potent HER3 Monoclonal Antibody That Blocks Both Ligand-Dependent and -Independent Activities: Differential Impacts of PTEN Status on Tumor Response.

HER3/ERBB3 is a kinase-deficient member of the EGFR family receptor tyrosine kinases (RTK) that is broadly expressed and activated in human cancers. HER3 is a compelling cancer target due to its important role in activation of the oncogenic PI3K/AKT pathway. It has also been demonstrated to confer tumor resistance to a variety of cancer therapies, especially targeted drugs against EGFR and HER2. HER3 can be activated by its ligand (heregulin/HRG), which induces HER3 heterodimerization with EGFR, HER2, or other RTKs. Alternatively, HER3 can be activated in a ligand-independent manner through heterodimerization with HER2 in HER2-amplified cells. We developed a fully human mAb against HER3 (KTN3379) that efficiently suppressed HER3 activity in both ligand-dependent and independent settings. Correspondingly, KTN3379 inhibited tumor growth in divergent tumor models driven by either ligand-dependent or independent mechanisms in vitro and in vivo Most intriguingly, while investigating the mechanistic underpinnings of tumor response to KTN3379, we discovered an interesting dichotomy in that PTEN loss, a frequently occurring oncogenic lesion in a broad range of cancer types, substantially blunted the tumor response in HER2-amplified cancer, but not in the ligand-driven cancer. To our knowledge, this represents the first study ascertaining the impact of PTEN loss on the antitumor efficacy of a HER3 mAb. KTN3379 is currently undergoing a phase Ib clinical trial in patients with advanced solid tumors. Our current study may help us optimize patient selection schemes for KTN3379 to maximize its clinical benefits. Mol Cancer Ther; 15(4); 689-701. ©2016 AACR.

Abstract LB-B11: Assessment of HER3 status during lapatinib treatment in HER3-positive breast cancer using 89Zr-anti-HER3 mAb

Abstract Treatment of human epidermal growth factor receptor 2 (HER2)-driven breast cancer with the HER-targeting tyrosine kinase inhibitor lapatinib can lead to a rapid compensatory increase in expression, signaling activity and relocalization of HER3 to the plasma membrane, which may attenuate the response to lapatinib. This might imply a potential role for a more dynamic assessment of HER3 tumor status using molecular imaging techniques, such as positron emission tomography (PET), instead of immunohistochemical HER3 staining on tumor biopsies. Here, we explored the feasibility of a dynamic assessment of HER3 status during lapatinib treatment in human breast cancer xenografts using zirconium-89 labeled anti-human HER3 monoclonal antibody (mAb) as a potential tracer for animal PET imaging. The anti-human HER3 mouse mAb MAB3481 was used for all experiments. The effect of lapatinib treatment on HER3 expression and HER3 mAb internalization in human breast cancer cell lines SKBR3 and BT474 was determined using flow cytometry. Biodistribution was performed using 89Zr-anti-HER3 mAb in mice bearing BT474 or SKBR3 tumors. Mice received daily vehicle or a lapatinib dose of 25, 50 or 100 mg/kg orally. A tracer dose of 89Zr-anti-HER3 mAb combined with the aspecific tracer 111In-IgG was injected 3 days after treatment. Ex vivo organ distribution assessment of 89Zr-anti-HER3 mAb was performed 6 days after tracer injection. Ex vivo tumor analysis using western blotting, ELISA and immunohistochemistry were performed to measure HER3 levels. In vitro, lapatinib treatment resulted in a ∼2-fold increase in membranous HER3 expression and HER3 internalization in SKBR3 and BT474 tumor cells. 89Zr-anti-HER3 mAb tumor uptake was significantly higher compared to 111In-IgG uptake in BT474 (P &amp;lt; 0.01), demonstrating HER3 specific tumor uptake. SKBR3 xenografts did not show HER3 specific uptake, which was likely caused by the poor viability of the tumors. HER3 upregulation was observed in BT474 xenografts after lapatinib treatment for 9 days. The enhanced HER3 expression was related to a 76 ± 9% increase in 89Zr-anti-HER3 mAb tumor uptake at 25 mg/kg and 92 ± 27% at 50 mg/kg lapatinib. Lapatinib at the highest concentration (100 mg/kg) strongly inhibited tumor growth and did not increase tumor uptake of the tracer. In conclusion, HER3-specific uptake of 89Zr-anti-HER3 mAb was shown in breast cancer xenografts. HER3 upregulation after lapatinib treatment was related to an enhanced 89Zr-anti-HER3 mAb uptake in these xenografts. These promising data warrant future dynamic assessment of HER3 status with 89Zr-anti-HER3 mAb PET imaging. Citation Format: Arjan Kol, Martin Pool, Steven de Jong, Elisabeth GE de Vries, Marjolijn N. Lub-de Hooge, Anton GT Terwisscha van Scheltinga. Assessment of HER3 status during lapatinib treatment in HER3-positive breast cancer using 89Zr-anti-HER3 mAb. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B11.

Immuno-PET Imaging of HER3 in a Model in which HER3 Signaling Plays a Critical Role

HER3 is overexpressed in various carcinomas including colorectal cancer (CRC), which is associated with poor prognosis, and is involved in the development of therapy resistance. Thus, an in vivo imaging technique is needed to evaluate the expression of HER3, an important therapeutic and diagnostic target. Here, we report successful HER3 PET imaging using a newly generated anti-human HER3 monoclonal antibody, Mab#58, and a mouse model of a HER3-overexpressing xenograft tumor. Furthermore, we assessed the role of HER3 signaling in CRC cancer tissue-originated spheroid (CTOS) and applied HER3 imaging to detect endogenous HER3 in CTOS-derived xenografts. Cell binding assays of 89Zr-labeled Mab#58 using the HER3-overexpressing cell line HER3/RH7777 demonstrated that [89Zr]Mab#58 specifically bound to HER3/RH7777 cells (Kd = 2.7 nM). In vivo biodistribution study in mice bearing HER3/RH7777 and its parent cell xenografts showed that tumor accumulation of [89Zr]Mab#58 in HER3/RH7777 xenografts was significantly higher than that in the control from day 1 to day 4, tending to increase from day 1 to day 4 and reaching 12.2 ± 4.5%ID/g. Radioactivity in other tissues, including the control xenograft, decreased or remained unchanged from day 1 to day 6. Positron emission tomography (PET) in the same model enabled clear visualization of HER3/RH7777 xenografts but not of RH7777 xenografts. CTOS growth assay and signaling assay revealed that CRC CTOS were dependent on HER3 signaling for their growth. In PET studies of mice bearing a CRC CTOS xenograft, the tumor was clearly visualized with [89Zr]Mab#58 but not with the 89Zr-labeled control antibody. Thus, tumor expression of HER3 was successfully visualized by PET with 89Zr-labeled anti-HER3 antibody in CTOS xenograft-bearing mice, a model that retains the properties of the patient tumor. Non-invasive targeting of HER3 by antibodies is feasible, and it is expected to be useful for cancer diagnosis and treatment.

Abstract LB-261: Mechanism of action of MCLA-128, a humanized bispecific IgG1 antibody targeting the HER2:HER3 heterodimer

Abstract Background: MCLA-128 is an ADCC-enhanced humanized common light chain bispecific IgG1 antibody that targets the HER2:HER3 dimer with nanomolar affinity, potently inhibiting tumor growth in vitro and in vivo. MCLA-128 shows superior activity to the combination trastuzumab/ pertuzumab and HER3 targeting monoclonal antibodies and is currently being evaluated in a Phase I clinical trial. This study investigated the mechanism of action of MCLA-128. Methods: Phosphorylation of HER receptors and downstream signaling molecules was studied in vitro and in vivo on HER2-amplified cancer cell lines by Pathscan arrays, luminex beads and Western blot analysis. Inhibition of MCLA-128 cell growth in combination with tyrosine kinase inhibitors and small molecules targeting the MAPK and PI3 kinase/Akt pathway was determined by proliferation inhibition and high content imaging assays. The potential effect of MCLA-128 on primary cardiomyocytes in the presence of Doxorubicin was analyzed by measuring ATP. Binding of MCLA-128 to a panel of cell lines in comparison to HER2 and HER3 antibodies was determined by FACS. Results: In contrast to other HER2 and HER3 targeted agents, only MCLA-128 inhibited phosphorylation of HER3 and downstream Akt and ERK in HER2 amplified cell lines cultured with high concentrations of heregulin in vitro. In xenograft studies, growth inhibition of the trastuzumab-resistant cell line JIMT-1 by MCLA-128 was correlated with reduced HER2:HER3 dimerization and a profound inhibition of the PI3K pathway. Synergistic growth inhibition in vitro was observed when tyrosine kinase inhibitors or inhibitors of the PI3K pathway were added to HER2 amplified cancer cells in the presence of MCLA-128. MCLA-128 did not show any evidence of cardiotoxicity in vitro in contrast to trastuzumab. MCLA-128 binds and coats breast cancer cell lines with differing levels of HER2 expression more efficiently in comparison to monospecific HER2 or HER3 monoclonal antibodies. Conclusions: The unique simultaneous targeting of MCLA-128 to HER2 and HER3 on HER2-overexpressing breast cancer cells leads to severe impairment of PI3K signaling and reduced cell growth whereas proliferation of primary cardiomyocytes is unaffected. The enhanced coating effect of MCLA-128 also supports its ADCC activity. Citation Format: Cecile Geuijen, Eric Rovers, Tristan Gallenne, David Maussang-Detaille, Arjen Kramer, Nellie Nieuwenhuizen, Carina Clements, Katinka van Zoest, Roy Nijhuis, Therese Visser, Renate Den Blanken-Smit, Willem Bartelink, Vanessa Zondag-van der Zande, Linda Kaldenberg, Pieter-Fokko van Loo, Rob Roovers, Robert Doornbos, Leo Price, Stefan Braam, Setareh van Driel, Lex Bakker, Ton Logtenberg, John de Kruif, Mark Throsby. Mechanism of action of MCLA-128, a humanized bispecific IgG1 antibody targeting the HER2:HER3 heterodimer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-261. doi:10.1158/1538-7445.AM2015-LB-261

Abstract 1558: Association of ErbB/HER biomarkers with antitumor activity of the anti-ErbB3/HER3 monoclonal antibody KTN3379 in SCCHN

Abstract Aberrant regulation of the HER family of receptor tyrosine kinases (RTK) has proven to be a driver for promoting tumor growth and progression in a number of solid tumors. HER3 is a unique RTK; it is a pseudo-kinase and requires heterodimerization with HER2 or other RTKs to signal. KTN3379 is a human anti-HER3 monoclonal antibody currently being evaluated in clinical studies. It potently inhibits HER3 by locking the receptor in an inactive conformation, providing a structural model for KTN3379 inhibition of both ligand-dependent and independent activation of HER3. Tumor expression of neuregulin 1 (NRG1), a high affinity HER3 ligand, is not well defined. An in silico survey of 29,049 human tumor samples, identified squamous cell carcinoma of the head and neck cancer (SCCHN) as having the highest prevalence of NRG1 over-expression. We used the VeraTag® technology, a proximity-dependent dual-antibody immunoassay, to quantify total receptor levels (HER1, HER2 and HER3) and dimerization/activation (HER1-HER1 homodimer, HER2-HER3 heterodimer, HER3-PI3 kinase (p85) and phospho-HER3) in formalin-fixed paraffin-embedded (FFPE) SSCHN tumors, and in cell lines and xenografts treated with KTN3379. In SSCHN tumors, expression of the HER biomarkers ranged from ∼6 to &amp;gt;300-fold, with the largest range in expression from HER1-HER1 homodimer and phospho-HER3, suggestive of signaling through both the HER1 and HER3 pathways in these tumors. The addition of KTN3379 to a panel of 6 SCCHN cell lines had no or a modest dose-dependent effect on cell growth, but resulted in a reduction of both phospho-HER3 and HER3-PI3K in 4 of the 6 SCCHN cell lines. Elevated levels of phospho-HER3 were predictive of KTN3379 activity. The combination of KTN3379 and cetuximab synergistically inhibited the growth of SCCHN cell lines greater than cetuximab alone, consistent with a reduction of pAKT and pERK by Western blot analyses. In mouse xenograft models, KTN3379 reduced tumor volume by 65% and 43% in Cal27 (SCCHN) and NCI-N87 (gastric), respectively. Tumor growth inhibition correlated with significant reductions in both phospho-HER3 (p = 0.0063) and HER3-PI3K (p = 0.0220) in the Cal27 model and phospho-HER3 (p = 0.0247) in the NCI-N87 model. In summary, we found that basal phospho-HER3 correlated with KTN3379 antitumor activity in SCCHN cell lines, and KTN3379 treatment of xenografts and cell lines resulted in a significant decrease of phospho-HER3 and HER3-PI3K suggesting phopho-HER3 may be useful as both a predictive and pharmacodynamic marker for KTN3379 activity. Collectively, these data support the evaluation of KTN3379 as an anti-tumor agent in NRG1-driven SCCHN cancer. A SCCHN window study evaluating the effects of KTN3379 in SCCHN cancer patients on phospho-HER3 and other biomarkers before and after treatment is planned. Citation Format: Diego Alvarado, Jerry Wallweber, Scott Seibel, Ahmed Chenna, Roy Ravanera, Weidong Huang, David Stathas, Paul Theobald, Theresa M. LaVallee. Association of ErbB/HER biomarkers with antitumor activity of the anti-ErbB3/HER3 monoclonal antibody KTN3379 in SCCHN. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1558. doi:10.1158/1538-7445.AM2015-1558

Time Trends of Left Ventricular Ejection Fraction and Myocardial Deformation Indices in a Cohort of Women with Breast Cancer Treated with Anthracyclines, Taxanes, and Trastuzumab

Trastuzumab, a HER2 monoclonal antibody, has transformed the prognosis of patients with the aggressive HER2-positive breast cancer type. Trastuzumab augments the cardiotoxic effects of anthracyclines, but its effect is thought to be at least partially reversible. The objective of this study was to examine the time trends of left ventricular (LV) size and function in a cohort of women treated with anthracyclines and trastuzumab. Twenty-nine patients >18years of age with first-time breast cancer treated with anthracyclines and trastuzumab were monitored using echocardiography before, at the completion of, and at a median follow-up of 24.7 months (interquartile range, 15.9-34 months) after the end of their cancer treatment. LV volume, LV ejection fraction, and global peak systolic longitudinal strain and strain rate were measured in the apical four- and two-chamber views. Left ventricular ejection fraction was measured using a modified Simpson's biplane method. LV end-diastolic and end-systolic volumes increased at the end of treatment compared with baseline and did not recover during follow-up. Left ventricular ejection fraction, strain, and strain rate decreased at the end of treatment compared with baseline (from 64±6% to 59±8%, from -20.0±2.5% to -17.6±2.6%, and from -1.26±0.23 to -1.13±0.16 sec(-1), respectively; P<.05 for all parameters) and remained decreased at follow-up. LV dilation and subclinical impairment in cardiac function persists >2years after the end of anthracycline and trastuzumab treatment, without significant recovery after trastuzumab cessation, suggestive of long-term underlying cardiac damage and remodeling.

Heregulin-HER3-HER2 signaling promotes matrix metalloproteinase-dependent blood-brain-barrier transendothelial migration of human breast cancer cell lines.

HER2-positive breast tumors are associated with a high risk of brain relapse. HER3 is thought to be an indispensible signaling substrate for HER2 (encoded by ERBB2) and is induced in breast cancer-brain metastases, though the molecular mechanisms by which this oncogenic dimer promotes the development of brain metastases are still elusive. We studied the effects of the HER3-HER2 ligand, heregulin (neuregulin-1, broadly expressed in the brain), on luminal breast cancer cell lines in vitro. Treatment of SKBr3 (ERBB2-amplified), MDA-MB-361 (ERBB2-amplified, metastatic brain tumor-derived) and MCF7 (HER2-positive, not ERBB2-amplified) cells with exogenous heregulin increased proliferation and adhesive potential, concomitant with induction of cyclin D1 and ICAM-1, and suppression of p27. All three cell lines invaded through matrigel toward a heregulin chemotactic signal in transwell experiments, associated with activation of extracellular cathepsin B and matrix metalloproteinase-9 (MMP-9). Moreover, heregulin induced breast cancer cell transmigration across a tight barrier of primary human brain microvascular endothelia. This was dependent on the activity of HER2, HER3 and MMPs, and was completely abrogated by combination HER2-HER3 blockade using Herceptin® and the humanized HER3 monoclonal antibody, EV20. Collectively these data suggest mechanisms by which the HER3-HER2 dimer promotes development of metastatic tumors in the heregulin-rich brain microenvironment.

Abstract 4661: The regulatory mechanism of fibronectin in EGFR and/or HER2 positive breast cancer

Abstract Fibronectin (FN) is a glycoprotein of the extracellular matrix and plays a major role in cell adhesion, migration and oncogenic transformation. Aberrant FN expression is associated with poor prognosis in a variety of cancers including breast cancer. However, the regulatory mechanism between FN and HER2 is not fully elucidated in breast cancer. Our clinical data showed that both disease free survival (DFS) and overall survival (OS) was lower in EGFR (+) and HER2 (+) breast cancer patients than in EGFR (+) breast cancer patients. To verify the causing of poor prognosis, we analyzed secreted proteins in EGFR and/or HER2 overexpressed MDA-MB231 cells. Our results showed that the level of FN expression was significantly increased in HER2 overexpressed MDA-MB231 cells. On the other hand, the induction of FN by HER2 overexpression was decreased by trastuzumab (HER2 monoclonal antibody) and AG825 (HER2 inhibitor), respectively. In addition, we observed that the expression of FN was dose-dependently increased by EGFR ligands in SKBR3 cells which are expressed endogenous EGFR and HER2. EGF ligands-induced FN expression also decreased through the suppression of HER2 in SKBR3 cell. However, these effects did not detect in trastuzumab-resistant breast cancer cell line, JIMT-1. Taken together, we demonstrated that the induction of FN by HER2 correlates poor clinical outcome in breast cancer patients. Citation Format: Myeongjin Jeon. The regulatory mechanism of fibronectin in EGFR and/or HER2 positive breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4661. doi:10.1158/1538-7445.AM2014-4661

Barriers to the Use of Trastuzumab for HER2+ Breast Cancer and the Potential Impact of Biosimilars: A Physician Survey in the United States and Emerging Markets.

Trastuzumab in combination with chemotherapy has become a standard of care for patients with HER2+ breast cancer. The cost of therapy, however, can limit patient access to trastuzumab in areas with limited financial resources for treatment reimbursement. This study examined access to trastuzumab and identified potential barriers to its use in the United States, Mexico, Turkey, Russia and Brazil via physician survey. The study also investigated if the availability of a biosimilar to trastuzumab would improve access to and use of HER2 monoclonal antibody therapy. Across all countries, a subset of oncologists reported barriers to the use of trastuzumab in a neoadjuvant, adjuvant or metastatic setting. Common barriers to the use of trastuzumab included issues related to insurance coverage, drug availability and cost to the patient. Overall, nearly half of oncologists reported that they would increase the use of HER2 monoclonal antibody therapy across all treatment settings if a lower cost biosimilar to trastuzumab were available. We conclude that the introduction of a biosimilar to trastuzumab may alleviate cost-related barriers to treatment and could increase patient access to HER2-directed therapy in all countries examined.

A phase 1 study of LJM716 in patients with esophageal squamous cell carcinoma, head and neck cancer, or HER2-overexpressing metastatic breast or gastric cancer.

2517 Background: The HER3 receptor is important for maintenance of EGFR- and HER2-driven cancers and targeted therapy resistance. LJM716 is a fully human HER3 monoclonal antibody active in ligand-d...

Barriers to the use of trastuzumab for HER2+ breast cancer and the potential impact of biosimilars: A physician survey in the United States and emerging markets.

610 Background: Biologics, such as trastuzumab, are an important part of oncology treatment paradigms, although access to such therapies is a challenge in emerging markets and in certain areas of more developed countries such as the United States (US). Biosimilars have the potential to increase access to biologic therapies by offering a comparable but more affordable alternative relative to the innovator biologic. This study examined access to trastuzumab and identified barriers to its use in HER2+ breast cancer patients in the US, Mexico (MEX), Turkey (TUR), Russia (RUS), and Brazil (BRZ). The study also examined whether availability of a biosimilar to trastuzumab would improve access to, and use of, HER2 monoclonal antibody therapy. Methods: Medical oncologists (N=500) completed a blinded survey examining their use of trastuzumab. Results: Across all countries, oncologists reported “not so often”, “rarely”, or “never” using trastuzumab in a neoadjuvant (27%), adjuvant (8%), or metastatic (8%) setting. C...

Genetic modification of T cells with a novel bispecific chimeric antigen receptor to enhance the control of high-grade glioma (HGG).

10027 Background: Highly targeted immunotherapy with adoptively transferred chimeric antigen receptor (CAR) T cells has shown considerable efficacy against some refractory childhood malignancies. Downregulation/mutation of targeted antigen can however create antigen loss escape variants. Multispecific T-cell approach could therefore further improve their therapeutic efficacy. Methods: We created a novel bispecific CAR that incorporates two extracellular antigen-recognition domains; one for HER2 (from HER2 monoclonal antibody FRP5) and the other for IL-13Rα2 (a mutated IL-13 molecule), in tandem (TanCAR). TanCAR was rationally designed by in silico modeling. Intracellular domain consists of a CD28/zeta-signaling chain. TanCAR encoding DNA was then synthesized and force expressed on T cells using retroviral system. Surface expression of extracellular domain of the TanCAR in its entirety was confirmed using HER2/IL-13Rα2 specific methods on flowcytometry. Functionality of TanCAR T cells was evaluated using s...

BRCAA1 antibody- and Her2 antibody-conjugated amphiphilic polymer engineered CdSe/ZnS quantum dots for targeted imaging of gastric cancer.

Successful development of safe and highly effective nanoprobes for targeted imaging of in vivo early gastric cancer is a great challenge. Herein, we choose the CdSe/ZnS (core-shell) quantum dots (QDs) as prototypical materials, synthesized one kind of a new amphiphilic polymer including dentate-like alkyl chains and multiple carboxyl groups, and then used the prepared amphiphilic polymer to modify QDs. The resultant amphiphilic polymer engineered QDs (PQDs) were conjugated with BRCAA1 and Her2 monoclonal antibody, and prepared BRCAA1 antibody- and Her2 antibody-conjugated QDs were used for in vitro MGC803 cell labeling and in vivo targeted imaging of gastric cancer cells. Results showed that the PQDs exhibited good water solubility, strong photoluminescence (PL) intensity, and good biocompatibility. BRCAA1 antibody- and Her2 antibody-conjugated QD nanoprobes successfully realized targeted imaging of in vivo gastric cancer MGC803 cells. In conclusion, BRCAA1 antibody- and Her2 antibody-conjugated PQDs have great potential in applications such as single cell labeling and in vivo tracking, and targeted imaging and therapeutic effects' evaluation of in vivo early gastric cancer cells in the near future.

HER2 monoclonal antibodies that do not interfere with receptor heterodimerization-mediated signaling induce effective internalization and represent valuable components for rational antibody-drug conjugate design

The human epidermal growth factor receptor (HER)2 provides an excellent target for selective delivery of cytotoxic drugs to tumor cells by antibody-drug conjugates (ADC) as has been clinically validated by ado-trastuzumab emtansine (KadcylaTM). While selecting a suitable antibody for an ADC approach often takes specificity and efficient antibody-target complex internalization into account, the characteristics of the optimal antibody candidate remain poorly understood. We studied a large panel of human HER2 antibodies to identify the characteristics that make them most suitable for an ADC approach. As a model toxin, amenable to in vitro high-throughput screening, we employed Pseudomonas exotoxin A (ETA’) fused to an anti-kappa light chain domain antibody. Cytotoxicity induced by HER2 antibodies, which were thus non-covalently linked to ETA’, was assessed for high and low HER2 expressing tumor cell lines and correlated with internalization and downmodulation of HER2 antibody-target complexes. Our results demonstrate that HER2 antibodies that do not inhibit heterodimerization of HER2 with related ErbB receptors internalize more efficiently and show greater ETA’-mediated cytotoxicity than antibodies that do inhibit such heterodimerization. Moreover, stimulation with ErbB ligand significantly enhanced ADC-mediated tumor kill by antibodies that do not inhibit HER2 heterodimerization. This suggests that the formation of HER2/ErbB-heterodimers enhances ADC internalization and subsequent killing of tumor cells. Our study indicates that selecting HER2 ADCs that allow piggybacking of HER2 onto other ErbB receptors provides an attractive strategy for increasing ADC delivery and tumor cell killing capacity to both high and low HER2 expressing tumor cells.

Generation of HER2 monoclonal antibodies using epitopes of a rabbit polyclonal antibody

One of the issues in using polyclonal antibodies is the limited amount of reagent available from an immunisation, leading to batch-to-batch variation and difficulties in obtaining the same antibody performance when the same antigen is re-immunised into several separate animals. This led to the development of hybridoma technology allowing, at least theoretically, for an unlimited production of a specific binder. Nevertheless, polyclonal antibodies are widely used in research and diagnostics and there exists a need for robust methods to convert a polyclonal antibody with good binding performance into a renewable monoclonal with identical or similar binding specificity. Here we have used precise information regarding the functional recognition sequence (epitope) of a rabbit polyclonal antibody with attractive binding characteristics as the basis for generation of a renewable mouse monoclonal antibody. First, the original protein fragment antigen was used for immunisation and generation of mouse hybridoma, without obtaining binders to the same epitope region. Instead a peptide designed using the functional epitope and structural information was synthesised and used for hybridoma production. Several of the monoclonal antibodies generated were found to have similar binding characteristics to those of the original polyclonal antibody. These monoclonal antibodies detected native HER2 on cell lines and were also able to stain HER2 in immunohistochemistry using xenografted mice, as well as human normal and cancer tissues.

An Antibody That Locks HER3 in the Inactive Conformation Inhibits Tumor Growth Driven by HER2 or Neuregulin

HER2/HER3 dimerization resulting from overexpression of HER2 or neuregulin (NRG1) in cancer leads to HER3-mediated oncogenic activation of phosphoinositide 3-kinase (PI3K) signaling. Although ligand-blocking HER3 antibodies inhibit NRG1-driven tumor growth, they are ineffective against HER2-driven tumor growth because HER2 activates HER3 in a ligand-independent manner. In this study, we describe a novel HER3 monoclonal antibody (LJM716) that can neutralize multiple modes of HER3 activation, making it a superior candidate for clinical translation as a therapeutic candidate. LJM716 was a potent inhibitor of HER3/AKT phosphorylation and proliferation in HER2-amplified and NRG1-expressing cancer cells, and it displayed single-agent efficacy in tumor xenograft models. Combining LJM716 with agents that target HER2 or EGFR produced synergistic antitumor activity in vitro and in vivo. In particular, combining LJM716 with trastuzumab produced a more potent inhibition of signaling and cell proliferation than trastuzumab/pertuzumab combinations with similar activity in vivo. To elucidate its mechanism of action, we solved the structure of LJM716 bound to HER3, finding that LJM716 bound to an epitope, within domains 2 and 4, that traps HER3 in an inactive conformation. Taken together, our findings establish that LJM716 possesses a novel mechanism of action that, in combination with HER2- or EGFR-targeted agents, may leverage their clinical efficacy in ErbB-driven cancers.

Abstract 5461: A HER3 antibody that blocks ligand-independent HER2-HER3 dimerization sensitizes to HER2 and PI3K inhibitors .

Abstract We hypothesized that complete inhibition of HER3 is required for the optimal action of HER2 or PI3K/Akt inhibitors against HER2+ tumors. The HER3 monoclonal antibody LJM716 (LJM) inhibits ligand-induced and ligand-independent dimerization of HER3. Treatment with LJM inhibited P-HER3 and P-Akt in a panel of HER2+ breast cancer cell lines. In BT474 and SKBR3 cells, LJM inhibited HER2-HER3 dimers as assessed by coprecipitation of HER2 with HER3 antibodies. Using a DUAL antibody proximity base assay, formalin-fixed sections of HER2+ xenografts treated with LJM showed a reduction of HER2-HER3 dimers and P-HER3 compared to untreated controls. Treatment with LJM alone also induced regression of established BT474 xenografts. Mice with BT474 xenografts were treated with lapatinib and trastuzumab (Lap/T), LJM and trastuzumab (LJM/T) or lapatinib, trastuzumab and LJM716 (Lap/T/LJM). After 3 weeks of treatment, mice from the 3 groups exhibited nearly complete tumor regression (volume &amp;lt;25 mm3). At 28 weeks of follow-up with no treatment, 8/12 mice in the Lap/T group and 5/7 mice in the LJM/T exhibited tumor regrowth (≥200 mm3), whereas only 2 of 15 mice in the Lap/T/LJM group did so (p=0.0046, Log-rank test). We expanded our studies to examine the combination of LJM716 with the p110α (PI3Kα) specific inhibitor BYL719 (BYL). LJM in combination with BYL inhibited growth in monolayer and 3-D Matrigel and S473 P-Akt in HER2+ breast cancer cells with mutations in the PI3K pathway better than either single agent. MDA-MB-453 xenografts regressed completely after 3 weeks of therapy with BYL/LJM whereas either single agent inhibited xenograft growth only partially. All mice bearing BT474 xenografts treated with T/LJM, T/BYL, LJM/BYL or T/LJM/BYL exhibited tumor regression after 24 days of treatment. Twenty weeks after treatment discontinuation, only 1/8 mice in the T/LJM/BYL exhibited tumor re-growth whereas &amp;gt;80% of mice in all other groups did so (p=0.0009, Log-rank test). Finally, we hypothesized that LJM would be at least equivalent to the HER2 antibody pertuzumab (P) in combination with T against HER2+ tumors. Inhibition of growth in vitro and P-Akt were superior with LJM/T compared to P/T in vitro. Mice bearing BT474 xenografts treated with either LJM/T or P/T had a similar nearly complete tumor regression after 4 weeks of treatment. We are currently monitoring potential tumor regrowth in these mice. In summary, treatment with LJM inhibited HER2-HER3 dimers, P-HER3 and P-Akt in HER2+ breast cancer cells with PI3K pathway mutations. As a single agent, LJM markedly inhibited HER2+ xenograft growth. Treatment with LJM in combination with Lap/T improved survival of mice with HER2+ xenografts compared to Lap/T. Further, LJM sensitized breast cancer cells and xenografts to a p110α-specific inhibitor. Finally, the HER3 antibody in combination with T inhibited PI3K/Akt and xenograft growth as well as the combination of P/T. Citation Format: Joan T. Garrett, Cammie R. Sutton, Carl Uli Bialucha, Seth A. Ettenberg, Jerry Wallweber, Lisa DeFazio-Eli, Carlos L. Arteaga. A HER3 antibody that blocks ligand-independent HER2-HER3 dimerization sensitizes to HER2 and PI3K inhibitors . [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 5461. doi:10.1158/1538-7445.AM2013-5461

MEK Inhibition Increases Lapatinib Sensitivity Via Modulation of FOXM1

The standard targeted therapy for HER2-overexpressing breast cancer is the HER2 monoclonal antibody, trastuzumab. Although effective, many patients eventually develop trastuzumab resistance. The dual EGFR/HER2 small molecule tyrosine kinase inhibitor lapatinib is approved for use in trastuzumab-refractory metastatic HER2-positive breast cancer. However, lapatinib resistance is a problem as most patients with trastuzumab-refractory disease do not benefit from lapatinib. Understanding the mechanisms underlying lapatinib resistance may ultimately facilitate development of new therapeutic strategies for HER2-overexpressing breast cancer. Our current results indicate that MEK inhibition increases lapatinib-mediated cytotoxicity in resistant HER2-overexpressing breast cancer cells. We genetically and pharmacologically blocked MEK/ERK signaling and evaluated lapatinib response by trypan blue exclusion, anchorage-independent growth assays, flow cytometric cell cycle and apoptosis analysis, and in tumor xenografts. Combined MEK inhibition and lapatinib treatment reduced phosphorylated ERK more than single agent treatment. In addition, Western blots, immunofluorescence, and immunohistochemistry demonstrated that the combination of MEK inhibitor plus lapatinib reduced nuclear expression of the MEK/ERK downstream proto-oncogene FOXM1. Genetic knockdown of MEK was tested for the ability to increase lapatinib-mediated cell cycle arrest or apoptosis in JIMT-1 and MDA361 cells. Finally, xenograft studies demonstrated that combined pharmacological inhibition of MEK plus lapatinib suppressed tumor growth and reduced expression of FOXM1 in HER2-overexpressing breast cancers that are resistant to trastuzumab and lapatinib. Our results suggest that FoxM1 contributes to lapatinib resistance downstream of MEK signaling, and supports further study of pharmacological MEK inhibition to improve response to lapatinib in HER2-overexpressing trastuzumab-resistant breast cancer.