HER2-targeted Monoclonal Antibody Research Articles

Anti-HER2 scFv Expression in Escherichia coli SHuffle®T7 Express Cells: Effects on Solubility and Biological Activity.

Breast cancer is the second most commonly diagnosed cancer, worldwide. Human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer is correlated with poor prognosis. HER2-targeting monoclonal antibodies resulted in longer survival of HER2+ breast cancer. Single-chain variable fragment (scFv) demonstrates improved penetrability into tumors. Due to the presence of two disulfide bond, scFv expression in reducing bacterial cytoplasm may cause formation of inclusion bodies. Disulfide bond can be formed properly in cytoplasm of SHuffle® strain as it is trxB-, gor-, and overexpresses cytoplasmic DsbC chaperone. In this study, the anti-HER2 scFv was successfully expressed and purified in BL21 (DE3) and SHuffle® cells. Here, significant higher soluble anti-HER2 scFv was produced in SHuffle® than in BL21 strain. The specific binding of anti-HER2 scFv to HER2 was shown by flow cytometry analysis and ELISA. Moreover, it was demonstrated that the anti-HER2 scFv produced in SHuffle® binds to HER2 at higher level as compared to that expressed in BL21 cells. Furthermore, competitive ELISA-based study suggested that anti-HER2 scFv recognizes the same epitope of HER2 receptor as the trastuzumab antibody. Our findings indicated that correct disulfide bond formation in SHuffle® strain can result in enhanced solubility and higher biological activity level of anti-HER2 scFv.

Abstract 3051: Targeting cdk12/13 re-sensitizes trastuzumab resistant her2+ breast cancers

Abstract Overexpression or amplification of the human epidermal growth factor receptor 2 (erbB2), which encodes for a receptor tyrosine kinase often referred to as HER2, occurs in about 20% breast cancer patients and is associated with aggressive disease and poor clinical outcome. The anti-HER2 therapy trastuzumab (a monoclonal antibody targeting HER2) has proven highly effective, increasing relapse free and overall survival for HER2 positive breast cancer patients. Nevertheless, acquisition of therapeutic resistance occurs frequently in advance disease. Recent studies have demonstrated that HER2 co-amplified genes may play a role in mechanisms of disease resistance. Cyclin dependent kinase 12 (CDK12) is among the genes that have been reported to be amplified in addition to HER2, and thus we aimed to investigate whether targeting CDK12 could be exploited as a therapeutic target and enhance and/or restore sensitivity in HER2 positive inhibitor resistant models of breast cancer. We have developed an in-house, highly selective, potent, and orally bioavailable CDK12/13 inhibitor, which effectively blocks proliferation and induces apoptosis in a panel of breast cancer cell lines and acts in synergy with trastuzumab in HER2 treatment resistant cell lines. Furthermore, our lead compound significantly slows tumor growth in trastuzumab resistant patient derived xenograft (PDX) models (as a single agent) and importantly, provokes greater efficacy in combination with trastuzumab. We are beginning to identify the signaling pathways underlying HER2 positive treatment resistance and how inhibition of CDK12/13 thwarts these resistance mechanisms. Accordingly, we anticipate that the development of CDK12/13 inhibitors holds promise as an anti-breast cancer strategy and future treatment option for HER2 positive breast cancer patients with recurrent disease. Citation Format: Francesca Vena, Max Aceti, Simon Bayle, Victor Quereda, Andrii Monastyrskyi, William Roush, Derek Duckett. Targeting cdk12/13 re-sensitizes trastuzumab resistant her2+ breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3051.

Advances in therapy of breast cancer: overexpression and therapeutic implications of targeting human epidermal growth factor receptors

The human epidermal growth factor receptor 2 (HER2) ERbb2 gene is amplified in approximately 25% of breast cancers. Characteristics of HER2-amplified tumors include increased proliferation rates and a propensity for distant metastasis. The discovery of overexpression of HER2 in a subset of breast cancers was an important milestone in our understanding of the biology of the disease. This paved the way for the discovery of trastuzumab, a humanized monoclonal antibody targeting HER2. Trastuzumab is the foundation of treatment of HER2- positive breast cancers, demonstrating dramatic responses in patients with metastatic disease. Recent advances in our understanding of the interaction between HER2 and other members of the epidermal growth factor receptor family have led to the identification of newer agents, resulting in the expansion of the clinical armamentarium of available agents for the treatment of HER2-positive tumors. The biology of the ERbb receptor family, the use of HER2-targeted agents in breast cancer, and the advances in anti-HER2 agents that are currently in clinical development are reviewed here.

Abstract P2-09-15: A phase I study of interferon-gamma (γ)plus weekly paclitaxel, trastuzumab and pertuzumab in patients with HER-2 positive breast cancer

Abstract Background: IFN-γ, a cytokine that plays diverse roles in innate and adaptive immunity, has been shown to be essential in anti-tumor immune response. In vitro and in vivo studies have shown the synergistic effect of IFN-γ in combination with HER2-targeting monoclonal antibodies with or without taxane chemotherapy. We have conducted a phase 1 clinical trial of systemic IFN-γ in combination with trastuzumab, pertuzumab, and paclitaxel in HER2-positive metastatic breast cancer. Methods: Two dose levels (DL) of IFN-γ, 50 (DL1) and 75 mcg/m2 (DL2), were evaluated. IFN-γ was given as subcutaneous injection three times weekly starting on day 1 of therapy for 12 weeks. Paclitaxel was administered intravenously (IV) weekly at 80mg/m2 in combination with trastuzumab IV (8 mg/kg loading dose, then 6 mg/m2 q 21 days) and pertuzumab IV (840 mg loading dose, then 420 mg q 21 days). Eligible patients had measurable metastatic HER2-positive breast cancer, were candidates to receive paclitaxel chemotherapy, and had an ECOG PS 0-1. The primary objective of this study was to evaluate the safety and tolerability of the combination therapy during the 12 weeks of treatment and to determine the recommended phase II dose (RP2D). Dose-limiting toxicity (DLT) during cycle one was defined as follows: Non-hematologic or hematologic toxicities that are ≥ grade 3 and probably or definitely related to study therapy which lead to chemotherapy treatment delays > 14 days. Results: A total of nine patients (3 on DL1 and 6 on DL2) were enrolled between 2/2017 and 11/2017. No DLT was observed. For DL1, no serious adverse events (SAE) or significant adverse events (AE) were observed among 3 patients who completed 12 weeks of treatment. For DL2, two out of 6 patients had SAEs including grade 3 pneumonitis (at week 8; treatment was subsequently discontinued) and grade 3 non-neutropenic fever (at week 6), which were possibly related to study treatment. These toxicities, however, did not meet the protocol definition of DLT. Based on these findings suggesting an improved tolerability of DL1 (50 mcg/m2), DL1 was selected as the RP2D. The most frequently observed grade 1 and 2 AEs that were at least possibly related to IFN-γ were fatigue (45%) nausea (36%), myalgia (36%), and fever (27%) diarrhea (18%). No grade 4 AE was noted. Grade 3 AEs included diarrhea, nausea, pneumonitis, non-neutropenic fever. Three out of 9 patients achieved partial response and 6 patients had stable disease per RECIST criteria. Conclusion: IFN-γ in combination with trastuzumab, pertuzumab, and paclitaxel was well tolerated in patients with HER2-positive metastatic breast cancer. Updated results will be presented and the phase 2 neoadjuvant trial is ongoing to further assess the efficacy of this approach. Citation Format: Han HS, Khong H, Costa R, Loftus L, Goodridge D, Henry T, Soliman H, Ismail-Khan R, Fridley B, Czerniecki B. A phase I study of interferon-gamma (γ)plus weekly paclitaxel, trastuzumab and pertuzumab in patients with HER-2 positive breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-09-15.

Cancer Cell Targeting With Functionalized Quantum Dot-Encoded Polyelectrolyte Microcapsules.

Imaging agents and drug carriers are commonly targeted toward cancer cell through functionalization with specific recognition molecules. Quantum dots (QDs) are fluorescent semiconductor nanocrystals whose extraordinary brightness and photostability make them attractive for direct fluorescent labeling of biomolecules or optical encoding of the membranes and cells. Here, we analyse the cytotoxicity of QD-encoded microcapsules, validate an approach to the activation of the microcapsule's surface for further functionalization with monoclonal antibody Trastuzumab, a humanized monoclonal antibody targeting the extracellular domain of the human epidermal growth factor receptor 2 (HER2) and already in clinical use for the treatment of HER2 positive breast cancer. In addition, we characterize the cell-specific targeting activity of the resultant bio-conjugate by immunofluorescence assay (IFA) and real-time analysis of interaction of the conjugates with live HER2 overexpressing human breast cancer cells. We demonstrate, that encapsulation of QDs into the polymer shell using the layer-by-layer deposition method yields highly fluorescent polyelectrolyte microcapsules with a homogeneous size distribution and biocompatibility upon in vitro treatment of cancer cells. Carbodiimide surface activation ensures optimal disperse and optical characteristics of the QD-encoded microcapsules before antibody conjugation. The prepared conjugates of the microcapsules with cancer-specific monoclonal antibody targeting HER2 provide sufficiently sensitive and specific antibody-mediated binding of the microcapsules with live cancer cells, which demonstrated their potential as prospective cancer cell–targeting agents.

Human epidermal growth factor receptor 2 amplification as a biomarker for treatment in patients with lymph node-metastatic penoscrotal extramammary Paget's disease.

The role of human epidermal growth factor receptor 2 (HER2) amplification as a biomarker for treatment in patients with lymph node (LN)-metastatic penoscrotal extramammary Paget's disease (EMPD) was investigated in the present study. A total of 11 male patients with LN-metastatic penoscrotal EMPD were retrospectively reviewed. Positron emission tomography/computed tomography (PET/CT) was conducted prior to surgery. Immunohistochemistry and fluorescence in situ hybridization were used to evaluate HER2 gene amplification in LN samples. Sanger sequencing was used to investigate HER2 mutations. A literature review of the prevalence of HER2 amplification in EMPD and the efficacy of HER2-targeted therapy was also undertaken. PET/CT is effective in detecting metastatic sites. The sensitivity and specificity of PET/CT was 90.9 and 100.0% for inguinal LNs, and 85.7 and 80.0% for pelvic LNs, respectively. The median time from LN dissection to disease progression was 15.9±1.5 months. Of the 11 patients, 3 (27.3%) indicated HER2 amplification. Patients with HER2 amplification showed shorter median times from disease discovery to LN metastasis (HER2 amplification vs HER2 non-amplification; 15.6 vs. 10.0 months; P=0.50) and from LN dissection to disease progression (HER2 amplification vs. HER2 non-amplification, 16.2 vs. 13.6 months; P=0.11). However, the aforementioned observations were not indicated to be statistically significant. No HER2 mutations were identified. Trastuzumab, a HER2-targeted monoclonal antibody, was administered to 2 of the patients with HER2 amplification. A literature review of the prevalence of HER2 amplification in EMPD and the efficacy of HER2-targeted therapy showed similar results. Altogether, 485 cases of EMPD were reported, 35 of which had metastases. The results in the present study suggest that PET/CT should be used on all metastatic EMPD patients. EMPD may be effectively treated with trastuzumab. The present study and case reports from the literature provide evidence for the benefit of testing for HER2 amplification in this rare disease and highlight the requirement for a multicenter clinical trial to assess the impact of trastuzumab therapy in treating this disease.

Population pharmacokinetic and covariate analyses of intravenous trastuzumab (Herceptin\xae), a HER2-targeted monoclonal antibody, in patients with a variety of solid tumors

PurposeThe aim of the study was to characterize the population pharmacokinetics (PK) of the intravenous formulation of trastuzumab, assess the impact of patient and pathological covariates on trastuzumab PK, and perform simulations to support dosing recommendations in special situations.MethodsSerum trastuzumab concentrations were obtained from 1582 patients with metastatic breast cancer (MBC), early breast cancer (EBC), advanced gastric cancer (AGC), or other tumor types/healthy volunteers in 18 phase I, II, and III trials and analyzed by nonlinear mixed-effects modeling.ResultsA two-compartment model with parallel linear and nonlinear elimination best described the data. During treatment, linear clearance (CL) dominated, resulting in a total CL of 0.173–0.337 L/day, which is similar to other IgG1 monoclonal antibodies. Covariates influencing CL were baseline body weight, aspartate aminotransferase, albumin, gastric cancer, and the presence of liver metastases. MBC and EBC had similar PK parameters, while CL was higher in AGC. Simulations indicated that at least 95% of patients with BC reach concentrations < 1 µg/mL (~ 97% washout) by 7 months. A dose delay in BC or AGC patients of > 1 week would take approximately 6 weeks to get back within steady-state exposure range.ConclusionsTrastuzumab PK for the intravenous formulation was well-described across cancer types, disease status, and regimens. No dose adjustment is required for any of the identified patient covariates. A 7-month serum washout period for trastuzumab is recommended. A reloading dose is required if a maintenance dose is missed by > 1 week.

Mechanisms Underlying the Action and Synergism of Trastuzumab and Pertuzumab in Targeting HER2-Positive Breast Cancer.

Human epidermal growth factor receptor (HER) 2 (HER2) is overexpressed in 20–30% of breast cancers. HER2 is a preferred target for treating HER2-positive breast cancer. Trastuzumab and pertuzumab are two HER2-targeted monoclonal antibodies approved by the Food and Drug Administration (FDA) to use as adjuvant therapy in combination with docetaxel to treat metastatic HER2-positive breast cancer. Adding the monoclonal antibodies to treatment regimen has changed the paradigm for treatment of HER2-positive breast cancer. Despite improving outcomes, the percentage of the patients who benefit from the treatment is still low. Continued research and development of novel agents and strategies of drug combinations is needed. A thorough understanding of the molecular mechanisms underlying the action and synergism of trastuzumab and pertuzumab is essential for moving forward to achieve high efficacy in treating HER2-positive breast cancer. This review examined and analyzed findings and hypotheses regarding the action and synergism of trastuzumab and pertuzumab and proposed a model of synergism based on available information.

PO-355 Antitumor activity of HSA-miR-X in HER2 positive cells is mediated by the regulation of AKT and ERK

IntroductionHER2 overexpression has been reported in approximately 30% of human breast tumours, and is associated with poor clinical outcome and recurrent disease. Herceptin is a humanised monoclonal antibody targeting HER2. PI3K-AKT is a downstream effector of the HER2 signalling pathway. microRNAs (miRNA) are small non-coding RNAs of 19–22 nucleotides that can negatively regulate gene expression at post-transcriptional levels. Herceptin plays an important role in treating metastatic breast cancer by targeting Erbb2 (HER2), therefore, combining Herceptin with miRNAs might enhance its antitumor activity. In this study the antitumor activity of hsa-miR-X, which was found to be Herceptin–responsive by our group, with Herceptin in HER2 positive breast cancer cell lines was identified in SK-Br-3 and BT-474 cells.Material and methodsWe investigated the synergistic effect of Herceptin and miRNA mimic combination on tumorigenic activities of the cells. To check its role in migration and invasion, wound healing and invasion assays were performed respectively. Cell invasion was monitored over a period of 24 hours by xCELLigence real-time cell analyzer measuring impedance-based signals. Furthermore, viability of breast cancer cells was measured by the WST1 assay after transfection with hsa-miR-X miRNA mimic. We measured the expression of AKT and ERK in HER2 positive cells treated with miR-X mimic plus Herceptin by Western blot analysis.Results and discussionsWe showed that increased expression of hsa-miR-X significantly inhibits migration and invasion of HER2 (+) breast cancer cells. Additionally, western blot experiments demonstrated that transfection of hsa-miR-X with Herceptin treatment decreased the protein level of AKT and ERK in these cells.ConclusionIt may be concluded that hsa-miR-X may increase the activity of the drug by pulling down its effective concentration and regulating PI3K-AKT pathway in HER2 positive cells. hsa-miR-X could decrease proliferation, motility and invasion via inhibiting Akt phosphorylation and may represent a new strategy for treating HER2 positive breast cancer.

An Intersection Between the Immune System and Cellular Signaling Pathways - The Impact of Anti-HER2 Monoclonal Antibodies on Individualized Approaches to Patients with Breast Cancer

1. Abstract Overexpression or gene amplification of Human Epidermal Growth Factor Receptor 2 (HER2) is present in approximately 20% of breast cancers, and indicates an aggressive tumor behavior. Activated HER homo and heterodimers orchestrate a downstream signaling network that regulates cell growth, proliferation and malignant spread. Trastuzumab, as a monoclonal antibody that inhibits the HER2, has immensely improved the outcome of patients with HER2-positive breast cancer. Since trastuzumab targets abnormal expression of HER2 in tumors, which are ‘addicted’ to HER2 activation, its therapeutic effects have been attributed to blocking of the intracellular signaling pathways. Moreover, the immune system is involved in the therapeutic impact of trastuzumab or similar anti-HER2 monoclonal antibodies. Various molecular pathways are relevant to HER2 overexpression, as well as the expression of other members of the Epithelial Growth Factor Receptor (EGRF) family. In addition, the estrogen and progesterone activity can interfere with the effects of trastuzumab and/or Chemotherapy (CHT) regimen, especially in patients with metastatic, HER2-positive malignancy. This article presents some insights to the signaling pathways, relevant to progression of breast cancer. It explains molecular characteristics that play a key role in affecting tumor response and resistance to HER2-targeted treatments. It also highlights the need for patient stratification, to develop the most reasonable de-escalation approaches, using HER2-targeted monoclonal antibodies. In addition, some practical strategies to overcome the resistance to trastuzumab, via targeting the main signaling pathways, in the context of HER2-positive and Hormone Receptor (HR)-positive metastatic or locally advanced breast cancer have been discussed, based on evidence from recent clinical trials. 2. Keywords: <span style=font-size:10.0pt;mso-ansi-language:EN-US;mso-fareast-language: PL>Breast Cancer; Estrogen Receptor (ER); Human Epidermal Growth Factor Receptor 2 (HER2) Monoclonal Antibodies; Trastuzumab; Signaling Pathways

Efficacy and Safety of Trastuzumab in Combination with S-1 and Cisplatin Therapy for Japanese Patients with HER2-Positive Advanced Gastric Cancer: Retrospective Analysis.

The combinations of oral fluoropyrimidines and cisplatin such as capecitabine and cisplatin (XP) or S-1 and cisplatin (SP) are regarded as a standard therapy against unresectable, recurrent, or advanced gastric cancer (AGC). Especially, SP is the most common regimen against AGC in Japan. For patients with human epidermal growth factor receptor type 2 (HER2)-positive AGC, trastuzumab, a monoclonal antibody targeting HER2 antibody, is additionally used in combination. Although trastuzumab in combination with XP (trastuzumab-XP) have been widely accepted, the efficacy of trastuzumab in combination with SP (trastuzumab-SP) lacks sufficient verification. The aim of the present study is to validate the comparability of trastuzumab-SP to trastuzumab-XP. Patients with HER2-positive AGC were assigned to the trastuzumab-XP or trastuzumab-SP group. We then retrospectively compared the efficacy and safety between both groups. As a first-line chemotherapy, trastuzumab in combination with XP or SP was administered to 58 patients: 28 with trastuzumab-XP and 30 with trastuzumab-SP. In the trastuzumab-XP group, response rate (RR), disease control rate (DCR), median progression-free survival (mPFS), and median overall survival (mOS) were 39.3%, 89.3%, 7.9 months, and 20.0 months, respectively. In the trastuzumab-SP group, RR, DCR, mPFS and mOS were 50.0%, 86.7%, 6.9 months, and 16.7 months, respectively. No significant difference in efficacy was observed between both groups. Severe hand-foot syndrome was observed more frequently in the trastuzumab-XP group than in the trastuzumab-SP group (14.3% vs. 0%, p = 0.05). Trastuzumab in combination with SP is a potential first-line therapeutic option for patients with HER2-positive AGC.

Localized co-delivery of collagenase and trastuzumab by thermosensitive hydrogels for enhanced antitumor efficacy in human breast xenograft

Modulation of the collagen-rich extracellular matrix (ECM) in solid tumors by the treatment with collagenase has been proved effective in enhancement of the interstitial transport and antitumor efficacy of antibodies. We, therefore, developed a PLGA-PEG-PLGA polymer-based thermosensitive hydrogel, which incorporated a HER2-targeted monoclonal antibody trastuzumab and collagenase (Col/Tra/Gel) for peritumoral administration. HER2-positvie BT474 tumor-bearing mice were selected as a model. The Col/Tra/Gel showed the continuous and biphasic release of protein drugs for 9 days in vitro. NIR imaging studies demonstrated a long-term retention of Col/Tra/Gel hydrogel in the peritumoral area for over 20 days. Treatment with Col/Tra/Gel reduced the collagen density and enhanced apoptotic cell death in tumor tissue, resulting in superior treatments with increased efficacy and reduced toxicity compared with other control groups. Moreover, a quarter-dose of Col/Tra/Gel exhibited a better antitumor efficacy than that of intravenous injection of clinical trastuzumab formulation. This localized co-delivery system offers a potential strategy for the modulation of dense ECM and enhancement of antibody efficacy.

From Natural Products to Designer Drugs: Development and Molecular Mechanisms Action of Novel Anti-Microtubule Breast Cancer Therapeutics.

Microtubule-targeted drugs (MTDs) have been on the forefront of breast cancer chemotherapy. Classic MTDs, such as paclitaxel and their semisynthetic derivatives, have achieved considerable success in the clinical management of breast neoplasms. In order to improve the specificity and to reduce undesirable, dose-limiting toxicities of these drugs, a plethora of novel compounds are being synthesized and investigated in laboratories worldwide. Due to their crucial roles during cell division, and to the fact that the suppression of their innate 'dynamic instability' can arrest cell cycle progression, microtubules formed an attractive target for cancer chemotherapy. Kadcyla (ado-trastuzumab emtansine), Halaven (eribulin mesylate), and Ixempra (Ixabepilone) are three relatively-novel, microtubule-targeting antibreast cancer drugs. Kadcyla was developed by conjugating a very potent derivative of the natural product maytansine to trastuzumab, a HER2-targeted monoclonal antibody. Kadcyla is a double-edged weapon, that is, it prevents receptor dimerization to inhibit cell proliferation, and then it enters inside the target tumour cell by receptor-mediated endocytosis and ensures death of the cell. Halaven (eribulin mesylate), created by simplifying the structure of the marine sponge-derived molecule Halichondrin B, works primarily by suppressing the growth rates of microtubules and thereby inducing cell cycle arrest and cell death. Ixabepilone, the semisynthetic analogue of epothilone B, suppresses the shortening rates of dynamic microtubules resulting in cell cycle inhibition and cell death. In order to improve the efficacy and reduce drug-induced side effects, novel therapeutic strategies, including liposome-mediated drug delivery, are being investigated.

Abstract CT162: Pre-specified interim analysis in a prospective, randomized phase II trial of trastuzumab vs trastuzumab + NeuVax to prevent breast cancer recurrence in HER2+ breast cancer patients

Abstract Introduction: The HER2-targeted monoclonal antibody, trastuzumab (Tz), is standard of care (SOC) for HER2-positive (HER2+) breast cancer (BCa) and has been shown to reduce recurrence. We have previously shown that NeuVax (E75 peptide + GM-CSF), a HER2-targeted peptide vaccine, is safe, immunogenic, and may have synergistic clinical efficacy when combined with Tz. Given the known cardiac toxicity of Tz, there is concern that adding a HER2-directed vaccine to Tz therapy may exacerbate this effect. We are currently enrolling patients (pts) in a multi-center, prospective, randomized, single-blinded, placebo-controlled phase II trial combining Tz and Neuvax in the adjuvant setting to prevent recurrence in HER2+ BCa pts. Here, we present the initial safety data. Methods: HLA-A2/A3+ BCa pts with stage I-III HER2+ disease at high risk for recurrence (pts not achieving complete response after Tz-containing neoadjuvant therapy or those undergoing up-front surgery with any node-positive disease if ER/PR- or ≥4 positive nodes if ER/PR+) were enrolled after SOC surgery, radiation and neo-adjuvant/adjuvant chemotherapy with approved Tz-containing regimen. Pts were randomized to receive Tz and NeuVax in the vaccine group (VG) or Tz and GM-CSF only in the control group (CG). Pts received vaccinations of NeuVax or GM-CSF intradermally every 3 weeks for 6 total vaccinations (primary vaccine series, PVS) starting with the third dose of Tz maintenance therapy. Starting 6 months after the completion of the PVS, pts received four booster inoculations, one every 6 months. Cardiac ejection fraction (EF) was assessed by either echo or MUGA at baseline and serially during treatment. Demographic and safety data were collected and analyzed. Safety analysis was initiated after enrollment of the 50th patient. Results: To date, we have enrolled 50 pts (VG n=22, CG n=28). There were no significant clinicopathologic differences between groups. There were no related grade 4 or 5 toxicities and no differences in related toxicities between the VG and CG (Grade 1: 96% vs 98.5%; Grade 2: 3.2% vs 1.5%; Grade 3: 0.8% vs 0%, p=0.14). There was no significant reduction in EF pre- to post-treatment in either group (VG: 61.1±5.4% vs 60.1±4.8%, p=0.55; CG: 62.3±5.7% vs 61.9±4.0%, p=0.74) and there was no difference in change between groups (p=0.54). Conclusion: The combination of Tz and Neuvax in HER2+ BCa pts is well tolerated and the cardiac effects from Tz are not worsened by the addition of NeuVax. We will continue to enroll up to our goal of 100 pts in this ongoing trial, and will report immunologic and clinical outcomes in the planned primary analysis after 24-months follow-up. Citation Format: Kaitlin M. Peace, Jennifer K. Litton, Rashmi Murthy, Timothy J. Vreeland, Diane F. Hale, Doreen O. Jackson, John S. Berry, Alfred F. Trappey, Garth S. Herbert, Guy T. Clifton, Mark O. Hardin, George E. Peoples, Elizabeth A. Mittendorf. Pre-specified interim analysis in a prospective, randomized phase II trial of trastuzumab vs trastuzumab + NeuVax to prevent breast cancer recurrence in HER2+ breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT162. doi:10.1158/1538-7445.AM2017-CT162

TMEM16A/ANO1 suppression improves response to antibody-mediated targeted therapy of EGFR and HER2/ERBB2.

TMEM16A, a Ca2+ -activated Cl- channel, contributes to tumor growth in breast cancer and head and neck squamous cell carcinoma (HNSCC). Here, we investigated whether TMEM16A influences the response to EGFR/HER family-targeting biological therapies. Inhibition of TMEM16A Cl- channel activity in breast cancer cells with HER2 amplification induced a loss of viability. Cells resistant to trastuzumab, a monoclonal antibody targeting HER2, showed an increase in TMEM16A expression and heightened sensitivity to Cl- channel inhibition. Treatment of HNSCC cells with cetuximab, a monoclonal antibody targeting EGFR, and simultaneous TMEM16A suppression led to a pronounced loss of viability. Biochemical analyses of cells subjected to TMEM16A inhibitors or expressing chloride-deficient forms of TMEM16A provide further evidence that TMEM16A channel function may play a role in regulating EGFR/HER2 signaling. These data demonstrate that TMEM16A regulates EGFR and HER2 in growth and survival pathways. Furthermore, in the absence of TMEM16A cotargeting, tumor cells may acquire resistance to EGFR/HER inhibitors. Finally, targeting TMEM16A improves response to biological therapies targeting EGFR/HER family members.

Biomarker analysis of the NeoSphere study: pertuzumab, trastuzumab, and docetaxel versus trastuzumab plus docetaxel, pertuzumab plus trastuzumab, or pertuzumab plus docetaxel for the neoadjuvant treatment of HER2-positive breast cancer

BackgroundNeoSphere showed significantly higher pathologic complete response (pCR) with neoadjuvant pertuzumab, trastuzumab, and docetaxel compared with trastuzumab plus docetaxel, pertuzumab plus trastuzumab, or pertuzumab plus docetaxel. We assessed associations between human epidermal growth factor receptor 2 (HER2) pathway-related biomarkers and clinical outcome in response to these regimens.MethodsTumor, serum, and whole blood samples were collected at baseline and post neoadjuvant treatment before surgery. Associations between biomarkers and pCR, and between biomarkers and clinical variables were assessed in the overall and estrogen receptor (ER)-positive and ER-negative populations. Changes in serum marker levels between baseline and post-neoadjuvant treatment were examined.ResultsNo markers were associated with pCR across all groups; however, significant associations were observed for two markers in individual groups. High HER2 was significantly associated with higher pCR rates (P = 0.001) and a significant treatment interaction (P = 0.0236) with pertuzumab, trastuzumab, and docetaxel (odds ratio 2.07, P = 0.01). Low serum transforming growth factor alpha (TGFα) was associated with higher pCR rates with pertuzumab plus trastuzumab (P = 0.04) without a significant treatment interaction. Presence of truncated HER2 did not affect pCR. A non-significant decreased pCR benefit was observed consistently across groups in patients with mutated PIK3CA while the treatment benefit from pertuzumab was maintained when comparing the trastuzumab plus docetaxel and pertuzumab, trastuzumab, and docetaxel groups. Notably, PIK3CA exon 9 mutations were associated with residual disease (pooled groups), which was not found for exon 20 mutations. Serum HER2 extracellular domain levels were significantly increased between baseline and post-neoadjuvant treatment in the non-trastuzumab-treated group, and decreased in the trastuzumab-containing groups (likely due to trastuzumab’s mechanism of action). Differences in biomarker profiles according to ER status were observed.ConclusionsThe observed associations of HER2 protein levels with sensitivity to pertuzumab, and of PIK3CA exon 9 mutation to lack of sensitivity to HER2-targeted monoclonal antibody treatment, warrant further investigation. Previously reported findings of truncated forms of HER2 as resistance markers to HER2-targeted treatment could not be confirmed in NeoSphere. Conventional HER2 assessment should continue and HER2 remains the only biomarker suitable for patient selection in this population.Trial registrationClinicaltrials.gov, NCT00545688. Registered on 16 October 2007.

Pre-clinical quantitative imaging and mouse-specific dosimetry for 111In-labelled radiotracers.

BackgroundAccurate quantification in molecular imaging is essential to improve the assessment of novel drugs and compare the radiobiological effects of therapeutic agents prior to in-human studies. The aim of this study was to investigate the challenges and feasibility of pre-clinical quantitative imaging and mouse-specific dosimetry of 111In-labelled radiotracers.Attenuation, scatter and partial volume effects were studied using phantom experiments, and an activity calibration curve was obtained for varying sphere sizes. Six SK-OV-3-tumour bearing mice were injected with 111In-labelled HER2-targeting monoclonal antibodies (mAbs) (range 5.58–8.52 MBq). Sequential SPECT imaging up to 197 h post-injection was performed using the Albira SPECT/PET/CT pre-clinical scanner. Mice were culled for quantitative analysis of biodistribution studies. The tumour activity, mass and percentage of injected activity per gram of tissue (%IA/g) were calculated at the final scan time point and compared to the values determined from the biodistribution data. Delivered 111In-labelled mAbs tumour absorbed doses were calculated using mouse-specific convolution dosimetry, and absorbed doses for 90Y-labelled mAbs were extrapolated under the assumptions of equivalent injected activities, biological half-lives and uptake distributions as for 111In.ResultsFor the sphere sizes investigated (volume 0.03–1.17 ml), the calibration factor varied by a factor of 3.7, whilst for the range of tumour masses in the mice (41–232 mg), the calibration factor changed by a factor of 2.5. Comparisons between the mice imaging and the biodistribution results showed a statistically significant correlation for the tumour activity (r = 0.999, P < 0.0001) and the tumour mass calculations (r = 0.977, P = 0.0008), whilst no correlation was found for the %IA/g (r = 0.521, P = 0.29). Median tumour-absorbed doses per injected activity of 52 cGy/MBq (range 36–69 cGy/MBq) and 649 cGy/MBq (range 441–950 cGy/MBq) were delivered by 111In-labelled mAbs and extrapolated for 90Y-labelled mAbs, respectively.ConclusionsThis study demonstrates the need for multidisciplinary efforts to standardise imaging and dosimetry protocols in pre-clinical imaging. Accurate image quantification can improve the calculation of the activity, %IA/g and absorbed dose. Diagnostic imaging could be used to estimate the injected activities required for therapeutic studies, potentially reducing the number of animals used.

Gastric Carcinoma at the Era of Targeted Therapies.

Gastric and gastro-esophageal cancers (GC/GEJ) appear as the second cancer-related death worldwide. Diagnosis is made at an advanced stage offering a curative attempt in less than 50% of cases. Despite the improvements of the systemic cytotoxic chemotherapy regimens, the prognosis of patients with metastatic GC/GEJ cancer remains poor. Recent insights in biochemical pathways have permitted to identify potential targets. The extracellular domain of HER2 receptors is implicated in cells' proliferation and in the anti-apoptotic process occurring in GC/GEJ cancers. Trastuzumab, a monoclonal antibody targeting HER2, in addition to chemotherapy permitted to obtain more than one year of survival in HER2-positive advanced GC/GEJ cancers. Recently, ramucirumab, a humanized monoclonal antibody targeting VEGFR-2 receptor demonstrated its efficacy as a second line treatment for patients with advanced GC/GEJ cancer. These encouraging results have justified evaluating targeted therapies in GC/GEJ cancers. In this review, we summarize targeted therapies that might present clinical efficacy in the treatment of advanced GC/GEJ cancers.

A novel mechanism of action of HER2 targeted immunotherapy is explained by inhibition of NRF2 function in ovarian cancer cells.

Nuclear erythroid related factor-2 (NRF2) is known to promote cancer therapeutic detoxification and crosstalk with growth promoting pathways. HER2 receptor tyrosine kinase is frequently overexpressed in cancers leading to uncontrolled receptor activation and signaling. A combination of HER2 targeting monoclonal antibodies shows greater anticancer efficacy than the single targeting antibodies, however, its mechanism of action is largely unclear. Here we report novel actions of anti-HER2 drugs, Trastuzumab and Pertuzumab, involving NRF2.HER2 targeting by antibodies inhibited growth in association with persistent generation of reactive oxygen species (ROS), glutathione (GSH) depletion, reduction in NRF2 levels and inhibition of NRF2 function in ovarian cancer cell lines. The combination of antibodies produced more potent effects than single antibody alone; downregulated NRF2 substrates by repressing the Antioxidant Response (AR) pathway with concomitant transcriptional inhibition of NRF2. We showed the antibody combination produced increased methylation at the NRF2 promoter consistent with repression of NRF2 antioxidant function, as HDAC and methylation inhibitors reversed such produced transcriptional effects. These findings demonstrate a novel mechanism and role for NRF2 in mediating the response of cancer cells to the combination of Trastuzumab and Pertuzumab and reinforce the importance of NRF2 in drug resistance and as a key anticancer target.

Pertuzumab and trastuzumab: the rationale way to synergy

It has now been 15 years since the HER2-targeted monoclonal antibody trastuzumab was introduced in clinical and revolutionized the treatment of HER2-positive breast cancer patients. Despite this achievement, most patients with HER2-positive metastatic breast cancer still show progression of their disease, highlighting the need for new therapies. The continuous interest in novel targeted agents led to the development of pertuzumab, the first in a new class of agents, the HER dimerization inhibitors. Pertuzumab is a novel recombinant humanized antibody directed against extracellular domain II of HER2 protein that is required for the heterodimerization of HER2 with other HER receptors, leading to the activation of downstream signalling pathways. Pertuzumab combined with trastuzumab plus docetaxel was approved for the first-line treatment of patients with HER2-positive metastatic breast cancer and is currently used as a standard of care in this indication. In the neoadjuvant setting, the drug was granted FDA-accelerated approval in 2013. Pertuzumab is also being evaluated in the adjuvant setting. The potential of pertuzumab relies in the dual complete blockade of the HER2/3 axis when administered with trastuzumab. This paper synthetises preclinical and clinical data on pertuzumab and highlights the mechanisms underlying the synergistic activity of the combination pertuzumab-trastuzumab which are essentially due to their complementary mode of action.