HER2 Antibody Research Articles

Tumor membrane vesicle (TMV)-based cancer vaccine induces dendritic cell maturation and downstream T cell activation

Abstract Breast cancer remains the most commonly diagnosed cancers among American women, with over 250,000 new cases expected per year. Breast cancer can be subdivided into different categories depending on the expression of receptors, most commonly into ER+, PR+, HER2+, and TNBC. Clinically, small molecule inhibitors of the hormone receptors and antibodies for HER2 have been developed to target these receptors and inhibit tumor signaling or eliminate these cells via phagocytosis and ADCC. Despite this, tumor cells that develop resistance to therapy remain a significant unmet need as they tend to be more aggressive and metastasize. We developed a novel immunotherapy approach that addresses the high degree of inter-patient and intra-tumor heterogeneity by making tumor membrane vesicle (TMV) vaccines out of individual tumor samples and modifying them with GPI-linked molecules like IL-12 and B7-1 to induce an antitumor immune response. In the present study, we investigated whether GPI-GM-CSF and GPI-IL-12 on TMVs from HER2+ murine breast cancer can target and activate antigen presenting cells. Here, we show that TMVs containing with GPI-GM-CSF and GPI-IL-12 can be phagocytosed by both macrophages and dendritic cells, induce upregulation of MHC-I, MHC-II and CD86, and inflammatory cytokine production in vitro. Further, when the DCs activated with our TMV vaccine were co-cultured with syngeneic T cells, they induced the upregulation of T cell activation markers. In summary, our results indicate that TMVs containing GPI-IL-12 and GPI-GM-CSF can enhance uptake and maturation of APCs and induce a strong T cell response downstream making it a potential therapeutic approach for HER2+ breast cancer.

High-speed Raman-encoded molecular imaging of freshly excised tissue surfaces with topically applied SERRS nanoparticles.

Surface-enhanced Raman scattering (SERS) nanoparticles (NPs) are increasingly being engineered for a variety of disease-detection and treatment applications. For example, we have previously developed a fiber-optic Raman-encoded molecular imaging (REMI) system for spectral imaging of biomarker-targeted SERS NPs topically applied on tissue surfaces to identify residual tumors at surgical margins. Although accurate tumor detection was achieved, the commercial SERS NPs used in our previous studies lacked the signal strength to enable high-speed imaging with high pixel counts (large fields of view and/or high spatial resolution), which limits their use for certain time-constrained clinical applications. As a solution, we explored the use of surface-enhanced resonant Raman scattering (SERRS) NPs to enhance imaging speeds. The SERRS NPs were synthesized de novo, and then conjugated to HER2 antibodies to achieve high binding affinity, as validated by flow cytometry. Under identical tissue-staining and imaging conditions, the targeted SERRS NPs enabled reliable identification of HER2-overexpressed tumor xenografts with 50-fold-enhanced imaging speed compared with our standard targeted SERS NPs. This enables our REMI system to image tissue surfaces at a rate of 150 cm2 per minute at a spatial resolution of 0.5mm.

A photosensitive liposome with NIR light triggered doxorubicin release as a combined photodynamic-chemo therapy system

The targeted drug delivery with the help of nanocarriers and the controlled drug release at the lesion sites are the most effective ways to enhance therapeutic efficacy and reduce side effects. Here, we built a light sensitive liposome (Her2-I&D-LSL) which was formed by a special phospholipid (PLsPC) and a hydrophobically modified photosensitizer (ICG-ODA). DOX was employed as the therapeutic drug, encapsulating in the internal phase of the liposome whose surface was modified by Her2 antibodies for recognizing tumor cells with high Her2 receptor expression. Mediated by NIR light, Her2-I&D-LSL was proved to generate sufficient ROS to realize PDT, which then triggered the release of DOX for combined chemotherapy. The ROS generation and DOX release were verified to be strictly controlled by NIR light and the proportion of ICG-ODA. Thanks to the mediation of Her2 receptor, the specific DOX release and the combination of PDT-chemotherapy triggered by NIR light, Her2-I&D-LSL showed a significant accumulation in MCF7 and SKOV3 tumors, thus leading to the strongest tumor growth inhibition effect compared to PDT alone (I-LSL) or chemotherapy alone (D-LSL). Her2-I&D-LSL also possessed a great biocompatibility due to the targeted treatment, holding promise for future cancer therapy in clinic.

Establishment of a Patient-derived Xenograft for Development of Personalized HER2-targeting Therapy in Gastric Cancer.

To maximize success rate for development of HER2-targeted therapeutics, patient-derived xenograft (PDX) models reflecting HER2-positive gastric cancer (HER2+ GC) patients were established. GC tissues obtained from surgery of GC patients were implanted into immune-deficient mice, and tumor tissue of HER2+ PDXs were verified of the patient-mimic HER2 expression by immunohistochemistry and explored for the feasibility by testing with Herceptin, the approved therapeutics and novel HER2 antibody therapeutics being developed. We obtained 5 cases of HER2+ GC PDX models reflecting patient's GC tumor, consisting of 2 cases of HER2 3+ and 2 cases of HER2 2+. Novel HER2 antibody displayed significantly improved anti-cancer efficacy in combination with Herceptin. The HER2+ GC PDX models were successfully established to be utilized for preclinical evaluation of HER2-targeting drugs and combined therapies for GC treatment, as an ideal platform of personalized tools for precision therapy.

Determination of HER-2/neu gene Status by Chromogenic in Situ Hybridisation Assay on Borderline (2+) Immunohistochemistry Cases in Patients with Invasive Breast Carcinoma: An Experimental Study on Preserved Tissue

HER-2 (also known as HER-2/neu) is a member of the epidermal growth factor (EGF) receptor family. The amplification of oncogene HER-2 is presented in 20 to 30% of breast cancers and results in an increase of the protein expression. HER-2 over expression has also been shown to correlate with poor prognosis. It is associated with poorly differentiated high grade tumors with lymph node involvement, greater risk of recurrence and relative resistance to some types of chemotherapy. The receptor is however a target for treatment with anti HER-2 antibody trastuzumab (Herceptin). Immunohistochemistry analysis for HER 2 scoring is subjective, requires trained personnel and expertise. One of the main concerns with IHC is that there is evidence of significant inter-observer variation in the assessment of staining, which can lead to misclassification of HER2 status. Chromogenic in situ hybridization (CISH) testing is sensitive and specific in detecting HER-2/neu gene amplification. Direct evaluation of gene amplification using CISH assay is a reliable method for routine diagnostic evaluation of HER2/neu status in breast cancer patients, especially in specimens showing 2+ IHC score. This observational experimental study was carried out in the Department of Pathology, Bangabandhu Sheikh Mujib Medical University, Dhaka, during the period of July 2014 to June 2015, The aim of this study was to assess HER-2 expression accurately in equivocal immunohistochemistry (2+) invasive breast cancer cases by Chromogenic in situ hybridization (CISH) and to associate the findings of CISH assay with the histologic prognostic and predictive factors (eg: tumor size, regional lymph node metastases, tumor grade and type. A total of 20 archival paraffin tissue blocks and IHC slides with IHC score 2+ for HER-2 were included in this study. All the slides were reviewed. CISH assay was done on section from archival paraffin block.CISH assay showed amplification of the HER-2 neu gene in 30% of cases. Majority (60%) was nonamplified. In two cases the results were unsatisfactory for interpretation. The differences were not statistically significant (p>0.05) among three groups (amplified, nonamplified and unsatisfactory) regarding the baseline characteristics (age and sex) with CISH. All the results of CISH assay in association with tumor size, tumor grade and lymphnode metastasis were not statistically significant. HER-2 / neu status is the one of the most important prognostic and predictive factor. It is a target for treatment with anti HER-2 antibody trastuzumab (Herceptin). Detection of HER-2/ neu status by IHC may sometimes be difficult and inaccurate, specially in IHC score 2+ cases. Based on the findings of the study, CISH can be considered a useful, simple and reproducible method for detecting HER-2/ neu gene amplification in cases with borderline (2+) immunohistochemistry score , and patients may be benefited from Trastuzumab therapy.

Immunogenicity of chimeric MUC1-HER2 vaccine against breast cancer in mice.

Objective(s):Breast cancer is one of the most common cancers in the world and is on the increase. MUC1 and HER2 as tumor-associated antigens (TAAs) are abnormally expressed to some extent in 75–80% of breast cancers. In our present research, a novel chimeric MUC1-HER2 (HM) protein was designed and used to study whether an immune response can be generated against these TAAs. In vitro analysis of the HER2-MUC1 construct confirmed the co-expression of MUC1 and HER2.Materials and Methods:BALB/c mice were immunized with this novel chimeric protein. The humoral immune response was assessed by enzyme-linked immunosorbent assay (ELISA). Then, BALB/c mice were injected subcutaneously 2×105 4T1-MUC1-HER2 tumor cells. Subsequently, tumor size and tumor necrosis measurements, MTT, cytokines assay and survival test were performed.Results:The results implied a critical role of HER2 and MUC1 antibodies in vaccination against breast cancer. This engineered protein can be a good vaccine to stop breast cancer.Conclusion:The results implied a critical role of HER2 and MUC1 antibodies in vaccination against breast cancer. This engineered protein can be a good vaccine to stop breast cancer.

Prognostic Value of Lymphangiogenesis Determinants in Luminal and Non-luminal Breast Carcinomas

Background:Breast carcinomas (BCs) are sub-classified according to the molecular characteristics into luminal and non-luminal subtypes that clinically show different biological behavior, treatment and prognosis. BCs spread primarily through lymphatic vessels using cascade processes of lymphagiogenesis in which VEGF-C plays an important role during lymph node metastasis. Prognostic value of VEGF-C in luminal and non-luminal BC is still unclear and has not been studied thoroughly to clarify and define prognosis and therapeutic monitoring.Aim:To define the prognostic value of lymphangiogenesis on survival rates of luminal and non-luminal subtypes BC.Materials and Methods:This study applied prospective cohort design, using 130 patients of invasive duct carcinoma of the breast, stage I-IIIA, from Sardjito General Hospital, Indonesia and subsequent longitudinal follow-up. Immunohistochemical staining was carried out using anti-ER, -PR, -Her-2, VEGF-C, VEGFR-3 and D2-40 antibodies. The related clinicopathologic characteristics of BC patients and lymphangiogenesis determinants, including VEGF-C expression, were statistically analyzed.Results:In non-luminal BC subtypes, VEGF-C expression (HR=0.04; 95% CI=0.01-0.41), lymph node metastasis (HR=0.14; 95% CI=0.04-0.55) and stage (HR=0.30; 95% CI= 0.02-0.76) were determined as independent prognostic factors on survival rates. However, the lymphangiogenesis determinants were not associated with the survival rates of luminal BC subtypes.Conclusion:This study suggested that lymphangiogenesis affects survival rates of non-Luminal subtype rather than the luminal subtypes of BC.

Immunohistochemical evaluation of insulin-like growth factor receptor 1 in breast cancer

Introduction/Objective. Activation of insulin-like growth factor receptor (IGF-1R) results in cell transition from growth phase to synthesis phase of cell cycle. Breast cancer is categorized into prognostic and therapeutic subtypes based upon hormone receptor, estrogen receptor (ER), and progesterone receptor (PR) expression and human epidermal growth factor receptor 2 (HER-2) expression. The objective of this study was to examine the expression of IGF-1R in ? specific subtype invasive breast cancer and its correlation with basic histopathological and immunohistochemical prognostic parameters. Methods. Formalin-fixed paraffin-embedded tumor samples were obtained from 129 female patients with invasive breast cancer (I?III disease stage) with the follow-up ranging 36?108 months (average 48 months). For immunohistochemical staining, we used monoclonal antibodies for ER, PR, IGF-1R, and polyclonal antibody for HER-2. Results. IGF-1R inversely correlated with tumor stage (p = 0.017), tumor grade (p = 0.001), HER-2 (p = 0.003), whereas significant positive correlation was found with multifocality/multicentricity of breast cancer (p = 0.036), ER (p = 0.001) and PR (p = 0.0001) expression. Cox-regression analysis for relapse-free survival (RFS) showed that disease stage (p = 0.039) and HER-2 (p = 0.033) were independent prognostic factors. IGF-1R did not predict clinical outcome in patients with breast cancer (p = 0.488, Kaplan?Meier test for RFS). Conclusion. Patients with low stage and grade hormone-dependent breast cancer had a significantly higher IGF-1R expression than patients with triple negative or HER-2 overexpressed cancer. The present findings also highlight that IGF-1R expression in multicentric/multifocal breast cancer supports the key roles in tumor initiation.

光纤生物传感器在HER3抗体药物定量检测中的应用

光纤生物传感器在HER3抗体药物定量检测中的应用

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.

CAIX furthers tumour progression in the hypoxic tumour microenvironment of esophageal carcinoma and is a possible therapeutic target

The hypoxic tumour microenvironment of solid tumours represents an important starting point for modulating progression and metastatic spread. Carbonic anhydrase IX (CAIX) is a known HIF-1α-dependent key player in maintaining cell pH conditions under hypoxia. We show that CAIX is strongly expressed in esophageal carcinoma tissues. We hypothesize that a moderate CAIX expression facilitates metastases and thereby worsens prognosis. Selective inhibition of CAIX by specific CAIX inhibitors and a CAIX knockdown effectively inhibit proliferation and migration in vitro. In the orthotopic esophageal carcinoma model, the humanized HER2 antibody trastuzumab down-regulates CAIX, possibly through CAIX’s linkage with HER2 in the hypoxic microenvironment. Our results show CAIX to be an essential part of the tumour microenvironment and a possible master regulator of tumour progression. This makes CAIX a highly effective and feasible therapeutic target for selective cancer treatment.

Prognostic Factors in Canine Mammary Carcinomas and HER2 Expression Relationship

Background: The human epidermal growth factor type 2 (HER2) receptor is a membrane glycoprotein tyrosine kinase. In woman, HER2 expression is diagnosed in 30% of breast carcinomas and it is associated with a worse prognosis, higher rate of recurrence and mortality. In the bitch, the HER2 overexpression in canine mammary tumors is still controversial and the prognostic value remains uncertain. Thus, we aimed to verify the HER2 expression in canine mammary carcinomas and relate it to the type and histological grade, lymph node metastasis and clinical staging.Materials, Methods & Results: Ninety bitches diagnosed with mammary carcinoma were included in this study. The inclusion criteria were bitches with complete clinical examination, thoracic radiographic examination and submitted unilateral or bilateral mastectomy. Ninety-nine samples of mammary carcinoma were used and the fragments of tumor and regional lymph nodes were fixed in 10% neutral formalin for histopathological and immunohistochemistry analysis. The lesions were evaluated by two pathologists and classified according to the type and histological grade. HER2 expression was performed by semi-quantitative analysis of the slides according to the HerceptTestTM (Dako) recommended score. Simple carcinomas were the most frequent (51.51%) followed by complex carcinomas (46.47%) and in situ carcinoma (2.02%). The histological grade of 97 carcinoma samples was attributed, except in situ carcinoma, 37 (38.14%) of the neoplasms were grade I, 50 (51.55%) grade II and only 10 (10.31%) tumors were classified as grade III. Forty bitches were submitted to clinical staging (TNM) and 42.50% of the bitches received staging in grade I and, 25% of the bitches staged in grade IV and V, with metastases. The HER2 expression, 13/99 samples (13.13%) received score +2, 19/99 (19.19%) score +1 and absence of marking (score 0) was identified in 67 samples (67.80 %). Immunostaining in hyperplastic or normal epithelial cells was evidenced, often in association with weak or moderate cytoplasmic labeling. Of the samples expressing +2 score for HER2 (n = 13), eight samples (17.39%) were complex carcinoma and five (9.80%) simple carcinomas. There was no relationship between HER2 immunostaining with age, tumor size, TNM, histological type, histological gradation, lymph node metastasis and distance. Animals with lymph node metastasis, as well as those diagnosed with distant metastasis, did not present HER2 expression in the tumors.Discussion: The simple carcinoma seems to be the most frequent type histological diagnosed in canine mammary carcinomas, followed by carcinoma in mixed tumor and complex carcinoma. Tubulopapillary carcinomas are more invasive in the female dogs as well as in the woman. Carcinomas grade I and II are more frequent and present a better prognosis for the dog. However, bitches with grade III carcinoma survived for a shorter time when compared to dogs with grade I or II tumors. A factor that may have contributed to the lower number of bitches at worst prognostic stage (EC IV and V) is the current owners’ awareness that they have sought veterinary help earlier, as soon as they detect small nodules in mammary gland. Overexpression of HER2 in women breast cancer is diagnosed in 20-30% of cases, whereas in bitches, this expression is variable. Also the different percentages of canine HER2 immunostaining are due to the lack of standardization for the analysis of the immunostaining, the immunohistochemical techniques employed and the non-specificity of the HER2 antibody. In canine mammary carcinomas the HER2 expression in low and this immunostaining is not related to other established prognostic factors. This study reinforces the hypothesis put forward by other authors that in the bitch the expression of HER2 may not be related to malignancy and tumor progression.

The Evolutionary Tale and Future Directions of Aromatase Inhibitors in Breast Carcinoma.

Aromatase inhibitors have often been likened to that of 'medical scalpels' for the treatment of breast carcinoma. By inhibiting the singular step of aromatisation, they have proven to be extremely effective allies in the treatment of breast cancer among postmenopausal women. However, their relevance soon may not be limited to the post-menopausal age group alone. Recent studies have hinted at their utility amongst the premenopausal women; combined with ovarian ablation techniques, aromatase inhibitors may prove to be equally effective and more, as compared to tamoxifen in this age-group. Additionally, explorations aimed at ascertaining their potential utility as an effective preventive strategy against breast carcinoma have yielded encouraging results. However, for aromatase inhibitors to be able to attain their full potential, further strategic fine-tuning aimed at maximising their efficacy and minimising their potentially far-reaching adverse effects, is the need of the hour. Despite the recent diversification, the issue of resistance to aromatase inhibitors in breast cancer threatens to derail the advances so gained till date. Fortunately, a few novel ploys have come to the fore, for instance combining aromatase inhibitors with HER-2 antibodies that could potentially help circumvent the menace of resistance in the near future. Till date, the utility of aromatase inhibitors can at best be described as onedimensional. However, with the unearthing of potential new avenues for its application, this assortment of molecules today stands on the precipice of ushering in a new revolution in the treatment of breast carcinoma.

NRG1-dependent activation of HER3 induces primary resistance to trastuzumab in HER2-overexpressing breast cancer cells.

This study was conducted to determine the role of neuregulin1 (NRG1)-dependent human epidermal growth factor receptor3 (HER3) activation in trastuzumab primary resistance, and to observe the inhibitory effect of HER3 monoclonal antibody on HER2-overexpressing breast cancer cells. BT474 cells (trastuzumab sensitive) and MDA-MB-453 cells (trastuzumab resistant) were first stimulated with NRG1 and then treated with either trastuzumab, HER3 antibody, or a combination of both. The expression of phospho human epidermal growth factor receptor2 (p-HER2), phospho human epidermal growth factor receptor3 (p-HER3), phospho protein kinaseB (p-Akt) and phospho mitogen-activated protein kinase (p-MAPK) were detected by western blotting. Apoptosis was detected by flow cytometry. Cell viability was detected by MTT assay. Without NRG1 stimulation, trastuzumab treatment significantly down-regulated the expression of p-HER2, increased early apoptosis, and decreased cell viability in BT474 cells. After NRG1 stimulation, the aforementioned effects weakened or disappeared in the trastuzumab treatment group, whereas in the HER3 antibody treatment group, there was significant downregulation in p-HER3 expression and increase in early apoptosis of BT474 cells. In MDA-MB-453cells, the HER3 antibody significantly downregulated both p-HER2 and p-HER3 and promoted early apoptosis after NRG1 stimulation, however, trastuzumab hardly played a role. p-Akt and p-MAPK were also significantly downregulated by the HER3 antibody after NRG1 stimulation. The expressions of p-HER2, p-HER3, p-Akt and p-MAPK were all downregulated after HER3 gene silencing, compared to the control. NRG1-dependent activation of HER3 induces primary resistance to trastuzumab in HER2-overexpressing breast cancer cells. HER3 monoclonal antibody combined with trastuzumab may serve as a treatment choice for patients with primary resistance to trastuzumab.

Combination of duligotuzumab, anti HER3 antibody or taselisib, PI3K INhibitor with trastuzumab shows synergetic antitumoral activity in HER2 positive gastric cancer cells

Introduction: The anti-HER2 monoclonal antibody trastuzumab is central to the treatment of HER2-positive gastric cancer (GC); however, its responses are limited due to some poorly understood mechanisms of resistance. The aim of this study was to asses the antitumortal activity of Duligotuzumab, an anti HER3 antibody or Taselisib, a Pi3k inhibitor combined with Trastuzumab in a panel of HER2 positive human gastric cancer cell lines (GCG), to improve anti HER2 treatment efficacy. Methods: We evaluated in vitro the effect of Duligotuzumab, Taselisib and Trastuzumabin single agent and in combination treatments in HER2-positive GCG (NCI-N87, KATOIII, OE19) and in negative HER2 GCC (MKN28), throughproliferation, migration and apoptosis assays. We also investigated the effect of combined treatment on downstream intracellular signaling, by western blot analysis. Results: After establishing, through a dose response curve, the IC50 for each drug used (≈ 0.5 µM), a significant synergistic effect of Duligotuzumab, Taselisib and Trastuzumab treatments in HER2-positive GCG was observed by reduction of proliferation and migration in KATOIII, OE19 and N87 cell lines; the same effect was found analyzing the apoptotic rate. At cellular level, in particular in KATOIII and OE19 cell lines, the combined treatment with Duligotuzumab or Taselisib plusTrastuzumabcompletely inhibited the activation of proteins downstream of HER3, PI3K and MAPK pathways. Conclusions: Targeting both HER2 and HER3 or HER2 and PI3K with the combination of anti-HE3 antibody or pi3k inhibitor with Trastuzumab may result in an improved treatment effects on HER2-positive GCG. These important findings can be utilized to facilitate the design of future clinical trials.

1641P - Combination of duligotuzumab, anti HER3 antibody or taselisib, Pi3k inhibitor with trastuzumab shows synergistic antitumoral activity in HER2 positive gastric cancer cells (GCC)

1641P - Combination of duligotuzumab, anti HER3 antibody or taselisib, Pi3k inhibitor with trastuzumab shows synergistic antitumoral activity in HER2 positive gastric cancer cells (GCC)

Surface modification of docetaxel nanocrystals with HER2 antibody to enhance cell growth inhibition in breast cancer cells

Here, we describe docetaxel nanocrystals (DTX-NCs) surface-modified with Herceptin® (HCT) for combination cancer therapy. Using nano-precipitation and adsorption method, DTX-NCs and HCT-DTX-NCs were successfully prepared. The morphologies of DTX-NCs and HCT-DTX-NCs were characterized by field emission scanning electron microscopy (FE-SEM). The particle size and surface charge were evaluated by zeta-sizer and zeta-potential, respectively. Moreover, DTX-NCs and HCT-DTX-NCs were evaluated in terms of an in vitro drug release, cellular uptake and cytotoxicity. We hypothesized that because they contain water-soluble HCT, HCT-DTX-NCs would have increased particle size and in vitro drug release, and well maintained stability compared with DTX-NCs. We also hypothesized that HCT-DTX-NCs would exhibit higher uptake by, and toxicity toward, HER2-receptor-bearing cells. The DTX-NCs and HCT-DTX-NCs exhibited pebble-shaped morphologies with mean sizes of 472.8±138.1nm and 526.4±130.4nm, respectively. The zeta-potentials of DTX-NCs and HCT-DTX-NCs were −9.6±2.81mV and +10.4±2.87mV, respectively. The drug release profiles from HCT-DTX-NCs were more rapid than those from DTX-NCs and DTX. Both the DTX-NCs and HCT-DTX-NCs exhibited good stability in PBS (pH 7.4) at 4°C for 4 weeks. Moreover, HCT-DTX-NCs were associated with significantly greater uptake by, and cytotoxicity toward, MCF-7 cells, than DTX-NCs and DTX. In conclusion, HCT-adsorbed DTX-NCs exhibited good cellular uptake and cytotoxicity and may be useful anti-cancer treatments.

Abstract 31: Preclinical development of a novel biparatopic HER2 antibody with activity in low to high HER2 expressing cancers

Abstract HER2-directed therapies have improved clinical outcomes for many patients with HER2 overexpressing breast and gastric cancer. Despite these successes, there remains a need to develop effective HER2-targeted therapies for these and other HER2-expressing tumors, particularly in the setting of recurrent or metastatic disease. One approach is to develop a single multifunctional antibody that has improved capacity and efficiency for binding HER2 compared with available HER2 inhibitors, can elicit ADCC, block ligand induced heterodimerization of HER2 with other HER family receptors, and down-regulate HER2 protein on the cell surface. ZW25 is a novel humanized, IgG1-like bispecific antibody directed against two distinct epitopes on the HER2 receptor. ZW25 has low nM binding affinity (0.9-16 nM) to recombinant HER2 and to cultured cancer cells with a range of HER2 expression, and shows higher maximal binding (1.4-1.9x) than monospecific HER2 antibodies on all tumor cell lines tested. The unique structure of ZW25 favors crosslinking of multiple HER2 receptors which is thought to promote clustering, and improves HER2 internalization compared to monospecific HER2 antibodies. In vitro studies demonstrated that ZW25 alone caused significant inhibition in growth of human cancer cell lines with a wide range of HER2 expression (5-54% inhibition depending on the cell line), and inhibited ligand-mediated tumor cell proliferation. ZW25 elicited concentration-dependent ADCC at nM potency (0.05-64 nM) with maximal lysis up to 52% on TNBC cell lines expressing HER2 at a 0/1+ level. ZW25 also exhibited synergy and additivity with multiple chemotherapeutic agents including platins, taxanes, microtubule inhibitors, and DNA synthesis inhibitors in various HER2 expressing tumor cell lines. In vivo studies demonstrated antitumor activity and/or improved survival against xenografts from a range of tumor types. In a HER2 3+ gastric cancer PDX model ZW25 induced significant tumor regressions, exhibiting 307% tumor growth inhibition relative to hIgG control and a 70% complete regression rate (trastuzumab exhibited 111% tumor growth control with no complete regressions). In a HER2 1+ breast cancer PDX model ZW25 significantly improved median survival (58 vs 28 days) and exhibited 141% tumor growth inhibition relative to hIgG control (trastuzumab exhibited 63% tumor growth inhibition, with median survival 39 days). In a low HER2-expressing NSCLC CDX model ZW25 improved median survival compared to hIgG control or cisplatin (> 66 vs 25 or 26 d respectively) and exhibited 159% tumor growth inhibition relative to hIgG control. In a GLP repeat dose toxicology study, ZW25 was well tolerated with no adverse effects at doses up to 150 mg/kg weekly IV for up to 8 weeks. Based on these findings, a first in human clinical trial of ZW25 has been initiated in patients with recurrent and/or metastatic HER2-expressing cancers. Citation Format: Nina Weisser, Grant Wickman, Rupert Davies, Gerry Rowse. Preclinical development of a novel biparatopic HER2 antibody with activity in low to high HER2 expressing cancers [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 31. doi:10.1158/1538-7445.AM2017-31

Extracellular Matrix/Integrin Signaling Promotes Resistance to Combined Inhibition of HER2 and PI3K in HER2+ Breast Cancer.

PIK3CA mutations are associated with resistance to HER2-targeted therapies. We previously showed that HER2+/PIK3CAH1047R transgenic mammary tumors are resistant to the HER2 antibodies trastuzumab and pertuzumab but respond to PI3K inhibitor buparlisib (TPB). In this study, we identified mechanisms of resistance to combined inhibition of HER2 and PI3K. TPB-resistant tumors were generated by treating HER2+/PIK3CAH1047R tumor-bearing mice long term with the drug combination. RNA sequencing of TPB-resistant tumors revealed that extracellular matrix and cell adhesion genes, including collagen II (Col2a1), were markedly upregulated, accompanied by activation of integrin β1/Src. Cells derived from drug-resistant tumors were sensitive to TBP when grown in vitro, but exhibited resistance when plated on collagen or when reintroduced into mice. Drug resistance was partially reversed by the collagen synthesis inhibitor ethyl-3,4-dihydroxybenzoate. Inhibition of integrin β1/Src blocked collagen-induced resistance to TPB and inhibited growth of drug-resistant tumors. High collagen II expression was associated with significantly lower clinical response to neoadjuvant anti-HER2 therapy in HER2+ breast cancer patients. Overall, these data suggest that upregulation of collagen/integrin/Src signaling contributes to resistance to combinatorial HER2 and PI3K inhibition. Cancer Res; 77(12); 3280-92. ©2017 AACR.

Peptide-Functionalized Nanomaterials for the Efficient Isolation of HER2-Positive Circulating Tumor Cells.

The detection of circulating tumor cells (CTCs) with a specific antigen expression is necessary in therapeutic response monitoring and targeted therapy guidance. The existence of human epidermal growth factor receptor 2 (HER2) and the concentration of HER2-positive CTCs are strong indicators for patient diagnosis, prognosis, and therapeutic monitoring. Herein we report the direct isolation of HER2-positive CTCs by peptide-functionalized nanomaterials. We designed and screened out a peptide as an HER2 antibody alternative demonstrating high HER2 affinity and selectivity. This HER2 recognition peptide bound efficiently with HER2 at the ligand-binding domain. Efficient HER2-positive CTC capture and detection were demonstrated using magnetic nanoparticles functionalized with the HER2 recognition peptide.