HER2-overexpressing Cancer Research Articles

Design and fabrication of cell-targeted, dual drug-loaded nanoparticles with pH-controlled drug release and near-infrared light-induced photothermal effects

Multi-functional nanoparticles with targeting and chemo-photothermal features are attractive materials for the development of therapeutics. In this study, thermal- and pH-sensitive nanoparticles loaded with two drugs were fabricated for controlled drug release. For synthesis of nanoparticles, we first prepared a PAMAM dendrimer cluster, which could provide large core space, and then sequentially loaded indocyanine green (ICG, a photothermal agent) and doxorubicin (DOX, a chemotherapeutic agent) premixed with polyacrylic acid (PAA) to the dendrimer cluster through ionic interactions. This self-assembling strategy yielded a high drug-loading content for both DOX (19.1 ± 0.4%) and ICG (54 ± 1%). Subsequently, PEG and Herceptin were covalently attached to nanoparticles to promote their bio-recognition by HER2-overexpressing cancer cells. DOX was released by changing pH from 7.4 to 5; DOX release was markedly enhanced by NIR (808 nm) laser irradiation. Conjugation with Herceptin could enhance specific attachment and accumulation of nanoparticles in tumor cells. Under NIR laser irradiation, ICG served as a remote-controlled trigger to generate considerable heat that caused severe physical damage and extensive cell apoptosis. Furthermore, after NIR laser irradiation, drug delivery and penetration of the nanoparticles into tumor spheroids were enhanced, indicating the potential therapeutic application of the newly developed nanoparticles.

Abstract 1773: Targeting HER2 and Src in HER2-driven ovarian and esophagogastric cancers

Abstract Background: Neratinib, an oral, irreversible pan-HER tyrosine kinase inhibitor, has exhibited clinical activity against HER2-mutant and HER2 over-expressing cancers. Our previous pre-clinical studies have shown that combining the multi-kinase inhibitor dasatinib with neratinib is highly synergistic in HER2-positive breast cancer. Therefore, the aim of this study was to examine the potential of combining neratinib and dasatinib in HER2 over-expressing/mutant esophagogastric (EG) and ovarian cancer. Methods: The prevalence of HER2 mutation and amplification levels in ovarian and EG cancers was determined using the AACR Project GENIE database. The anti-proliferative effects of neratinib and dasatinib were assessed by acid phosphatase assay in a panel of HER2-altered ovarian (Caov3, OVCAR8, IGROV-1) and EG (NCI-N87, OE19) cancers. Synergy was assessed using Combenefit software with the Loewe additivity model, with additivity and synergy > 0 and antagonism < 0. Western blotting was used to examine the basal expression of HER2 and downstream signalling activity and to investigate the signalling effects of drug treatment in HER2-mutant OVCAR8 cells. Results: HER2 was the most commonly amplified gene in EG cancers (14.1%) and was also amplified in 2.4% of ovarian cancers. HER2 mutations occurred in 6.1% of EG and 1.5% ovarian cancers. All five cell lines tested displayed nanomolar sensitivity to single agent neratinib or dasatinib (Table). The combination of neratinib and dasatinib was synergistic in all cell lines. The greatest synergy was observed with low concentrations of neratinib in EG cell lines. Synergistic interactions occurred across a range of concentrations with both drugs in ovarian cancer cell lines. Antagonism was not observed at any concentration tested. Neratinib plus dasatinib inhibited Src and MAPK activity in OVCAR8 cells. Neratinib and dasatinib in ovarian and EG cancersCell linesCancertypeNeratinib EC50 value (nM)Dasatinib EC50 value (nM)Combination effectNCI-N87Esophagogastric0.188979SynergismOE19Esophagogastric0.432284SynergismOVCAR8Ovarian16281SynergismIGROV-1Ovarian1706SynergismCaov3Ovarian13730Synergism Conclusions: These data suggest that the addition of dasatinib may enhance the anti-cancer effect of neratinib in HER2-driven ovarian and EG cancers and warrants further investigation. Citation Format: Neil T. Conlon, Keshia Kroh, John Crown, Denis M. Collins. Targeting HER2 and Src in HER2-driven ovarian and esophagogastric cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1773.

A phase II study of efficacy and safety of RC48-ADC in patients with locally advanced or metastatic HER2-overexpressing gastric or gastroesophageal junction cancers.

4560 Background: RC48-ADC is an antibody-drug conjugate (ADC) drug comprised of a novel humanized anti-HER2 IgG1, a linker, and a microtubule inhibitor, MMAE. The MoA included inhibition of HER2 signal pathway and cytotoxicity of MMAE. RC48-ADC has demonstrated promising anti-tumor activity in pre-clinical and early clinical studies. The current study is designed to evaluate the efficacy and safety of RC48-ADC in heavily treated patients with HER2-overexpressing (IHC 2+ or 3+) gastric or gastro-esophageal junction cancers. Methods: This is an open-label, multicenter, single-arm, phase II study. Eligibility criteria include: histologically confirmed gastric or gastro-esophageal junction cancers, HER2-overexpression (IHC 2+ or 3+), ECOG PS 0-1, post-to ≥2 prior systemic treatment. The patients received RC48-ADC, 2.5 mg/kg, q2w until disease progression, unacceptable toxicity, withdrawal, or study termination. The primary endpoint was ORR. PFS, OS, and safety were also evaluated. Results: Patient enrollment started in July 2017, and completed in November 2019. By the data cut-off date on 17-Dec-2019, 127 patients were enrolled. The median age was 58 years. At baseline, 59 patients (46.5%) had received ≥ 3 lines prior treatment. For the overall 127 patients, the investigator-assessed confirmed ORR was 18.1% (95% CI: 11.8%, 25.9%). Sub-group ORR was 19.4% and 16.9% for the patients post to 2 lines and ≥ 3 lines, respectively. For the 111 patients who were monitored for ≥ 2 cycles of efficacy assessments (i.e. 12 weeks), the ORR was 20.7% (95% CI: 13.6%, 29.5%). For the 127 patients, the mPFS was 3.8 months (95% CI: 2.7, 4.0, 89 events [70.1%]) and the mOS was 7.6 months (95% CI: 6.6, 9.2, 52 events [40.9%]). The most commonly reported treatment-related AEs were leukopenia (52.0%), alopecia (51.2%), neutropenia (48.0%), and fatigue (42.5%). Conclusions: RC48-ADC demonstrated a clinically meaningful response and survival benefit in the heavily treated patients with HER2-overexpressing gastric or gastro-esophageal junction cancers. The safety profile was in line with the previously reported data of RC48-ADC. RC48-ADC showed positive benefit/risk ratio for the target population. Clinical trial information: NCT03556345 .

Identifying host-intrinsic resistance mechanisms to therapeutic anti-tumor antibody

Abstract Monoclonal antibody (mAb) therapies have greatly improved outcomes for many cancer subtypes, many of which target tumor-associated antigens (TAA). Trastuzumab targets the TAA human epidermal growth factor 2 (HER2), inducing antibody dependent cellular cytotoxicity and phagocytosis (ADCC/ADCP) as well as interfering with receptor signaling. Despite its reputation as the gold standard treatment for HER2-overexpressing cancers, response rates remain low in metastatic breast cancer (≤30% as a monotherapy, ≤50% with chemotherapy). Multiple studies suggest induction of endogenous anti-tumor immunity is a determinant of therapeutic success. Utilizing the genetically heterogeneous Diversity Outbred (DO) mouse model, we seek to identify host-intrinsic mechanisms of resistance to TAA-mediated immunotherapy. We find a divergent response to αNeu (HER2 homolog) mAb clone 7.16.4 in (DOxBALB/c)F1 mice bearing established BALB/c-syngeneic TUBO mammary carcinoma. We find host genetic background regulates anti-tumor immune activity. ~25% showed complete response (CR) to therapy, ~60% a partial response (PR), and ~15% failed to respond (NR). CR mice were protected from a contralateral rechallenge, suggesting adaptive anti-tumor immunity was promoted by mAb therapy. Peripheral anti-tumor IgG, measured by flow cytometry, and IFNγ producing T cells, measured via ELISpot, also trend toward CR>PR>NR. Genetic linkage analysis using R/qtl2 identified loci associated with robust response to mAb therapy. Genes within these loci may represent potential routes of ingress toward addressing innate and acquired resistance during anti-TAA mAb therapy, particularly as regulators of immunogenicity of ADCC and ADCP. NIH R37 CA220482.

Protein-based drug carrier specific for HER2-positive breast cancer cells

The aim of this study was to design a protein-based single chain vehicle that be able to recognize and enter HER2-overexpressing cancer cells and subsequently enter their nuclei. To this end, we conjugated VH domain of an anti-HER2 antibody (Herceptin) as HER2 targeting element, nine arginine residues (summarily called 9R) as cell penetrating peptide (CPP), and nuclear localization signals (NLSs) of TUS and c-Myc proteins as nuclear delivery mediators. The recombinant construct was labeled with FITC fluorescent dye to trace within the cells. Fluorescent imaging revealed that the construct was able to specifically recognize HER2-overexpressing cells and enter their nuclei.

A novel anti-HER2 antibody GB235 reverses Trastuzumab resistance in HER2-expressing tumor cells in vitro and in vivo

HER2 overexpression is frequently associated with tumor metastasis and poor prognosis of breast cancer. More evidence indicates that HER3 is involved in HER2-resistant therapies. Combination treatments with two or more different monoclonal antibodies are a promising strategy to overcome resistance to HER2 therapies. We presented a novel fully human HER2-targeted monoclonal antibody, GB235, screened from a phage-display library against the HER2 antigen. GB235 in combination with Trastuzumab overcomes resistance in HER2-positive tumors and results in more sustained inhibition of tumor growth over time. The competition binding assay showed that the epitopes of GB235 do not overlap with those of Pertuzumab and Trastuzumab on HER2. Further HER2 mutagenesis results revealed that the binding epitopes of GB235 were located in the domain III of HER2. The mechanism of action of GB235 in blocking HER2-driven tumors is different from the mechanisms of Trastuzumab or Pertuzumab. GB235 does not affect the heterodimerization of HER2 and HER3, whereas the GB235 combined treatment with Trastuzumab significantly inhibited heregulin-induced HER3 phosphorylation and downstream signaling. Moreover, GB235 in combination with Trastuzumab reversed the resistance to heregulin-induced proliferation in HER2-overexpressing cancer cell lines. GB235 combined with Trastuzumab treatment in xenograft models resulted in improved antitumor activity. Complete tumor suppression was observed in the HER2-positive NCI-N87 xenograft model treated with the combination treatment with GB235 and Trastuzumab. In a Trastuzumab-resistant patient-derived tumor xenograft model GA0060, GB235 plus Trastuzumab reversed the resistance to Trastuzumab monotherapy. Because GB235 showed a different working mechanism with Pertuzumab and Trastuzumab, these agents can be considered complementary therapy against HER2 overexpression tumors.

Abstract P1-18-26: Risk of reduction of left ventricular ejection fraction with trastuzumab emtansine: A systematic review and meta-analysis of randomized controlled trials

Abstract Background: HER2 targeted therapies have significantly improved the prognosis of HER2 overexpressing cancers, in particular HER2-positive breast cancer. However, type-II cancer therapeutics-related cardiac dysfunction (CTRCD) characterized by a drop in left ventricular ejection fraction (LVEF) remains a significant adverse event for trastuszumab and other HER2 targeted therapies. Trastuzumab Emtansine (T-DM1) is a HER2 targeted antibody-drug conjugate consisting of anti-HER2 IgG1 antibody trastuzumab and a maytansine derivative microtubule inhibitor DM-1. Currently T-DM1 is approved for HER2-positive breast cancer both in the metastatic and in the adjuvant settings. We conducted a systematic review and meta-analysis of published phase 3 randomized controlled trials (RCTs) to determine the relative risk of reduction of LVEF with T-DM1 in comparison to other therapies in HER2-postive cancers. Methods: We conducted a systematic search at PUBMED, MEDLINE, EMBASE, and professional societies meeting abstracts as per PRISMA guidelines from inception until March 31st, 2019. Published phase 3 RCTs comparing T-DM1 with other therapies in patients with HER2-positive cancers and reporting the number of events of reduction in LVEF in both intervention and control arms were included in the final analysis. The primary meta-analytic approach was a random effects model using the Mantel-Haenszel (MH) method, and it was used to calculate the estimated pooled risk ratio (RR) with 95% confidence interval (CI). Heterogeneity was tested with I2 value and Cochran’s Q-test. Results: Five RCTs (EMILIA, GATSBY, KATHERINE, KRISTINE, and TH3RESA) randomizing 3802 patients (2080 in intervention arms and 1722 in control arms) with HER2-positive cancers were included in the final analysis. Four studies (EMILIA, KATHERINE, KRISTINE, and TH3RESA) were conducted in breast cancer patients, and one (GATSBY) was done in gastric/gastro-esophageal adenocarcinoma patients. GATSBY was a phase 2/3 study and rest were phase 3 RCTs. Patients in control arms received variable systemic therapies across trials, most of the studies used conventional HER2 targeting drugs (e.g. trastuzumab, lapatinib etc) with exception of GATSBY trial which only used taxanes. Dose of T-DM1 was usually 3.6 mg/kg every 3 weeks, except for GATSBY study where dose was 2.4 mg/kg every week. The pooled RR of any-grade reduction of LVEF was significantly lower in the T-DM1 group as compared to control at 0.58 (95% CI: 0.36-0.95, p = 0.03, I2 = 0). No publication bias was appreciated across all studies included in the final analysis. Conclusion: In our meta-analysis, the relative risk of reduction of LVEF was significantly lower in the T-DM1 containing regimens compared to other therapies in patients with HER2-positive cancers. It could be an area of research to identify the mechanism and to determine the clinical utility of this finding. Citation Format: Nusrat Jahan, Somedeb Ball, Lukman Tijani, Fred Hardwicke, Catherine Jones. Risk of reduction of left ventricular ejection fraction with trastuzumab emtansine: A systematic review and meta-analysis of randomized controlled trials [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-18-26.

Design Strategy to Create Antibody Mimetics Harbouring Immobilised Complementarity Determining Region Peptides for Practical Use

Monoclonal antibodies (mAbs) are attractive therapeutics for treating a wide range of human disorders, and bind to the antigen through their complementarity-determining regions (CDRs). Small stable proteins containing structurally retained CDRs are promising alternatives to mAbs. In this report, we present a method to create such proteins, named fluctuation-regulated affinity proteins (FLAPs). Thirteen graft acceptor (GA) sites that efficiently immobilise the grafted peptide structure were initially selected from six small protein scaffolds by computational identification. Five CDR peptides extracted by binding energy calculations from mAbs against breast cancer marker human epithelial growth factor receptor type 2 (HER2) were then grafted to the selected scaffolds. The combination of five CDR peptides and 13 GA sites in six scaffolds revealed that three of the 65 combinations showed specific binding to HER2 with dissociation constants (KD) of 270–350 nM in biolayer interferometry and 24–65 nM in ELISA. The FLAPs specifically detected HER2-overexpressing cancer cells. Thus, the present strategy is a promising and practical method for developing small antibody mimetics.

A new anti-HER2 antibody that enhances the anti-tumor efficacy of trastuzumab and pertuzumab with a distinct mechanism of action

The majority of patients with metastatic breast cancer who are treated with the anti-HER2 monoclonal antibody, trastuzumab, generally develop resistance to the drug within a year after initiation of the treatment. Here we describe a new anti-HER2 humanized monoclonal antibody, 19H6-Hu, which binds to HER2 extracellular domain (ECD) with high affinity and inhibits proliferation of multiple HER2-overexpressing cancer cell lines as a single agent or in combination with trastuzumab. 19H6-Hu binds to the domain III in proximity to the domain IV of HER2 ECD, which differs from trastuzumab and pertuzumab. 19H6-Hu in combination with trastuzumab was more effective at blocking phosphorylation of ERK1/2, AKT(S473)and HER2 (Y1248) in HER2-positive cancer cells compared to trastuzumab alone or in combination with pertuzumab. Combination of three antibodies, 19H6-Hu, inetetamab (a trastuzumab analog) and pertuzumab exhibited much stronger inhibition of large NCI-N87 tumor xenografts (>400mm3) than the current standard of care, inetetamab (trastuzumab) plus Docetaxel (DTX), as well as the combination of 19H6-Hu, inetetamab and DTX. Our results highlight the functional variability of HER2 domains and provide a new insight into the design of HER2-targeting agents.

Single-domain antibody C7b for address delivery of nanoparticles to HER2-positive cancers.

Monoclonal antibodies (mAb) demonstrate great potential as immunotherapy agents for the treatment of diseases such as cancer as well as tagging for the targeted delivery of multicomponent therapeutic or diagnostic systems. Nevertheless, the large physical size, poor stability of mAbs and abnormal allergic reactions still remain the main issues affecting their generalised use. Single-domain antibodies (sdAb) are seen as the next generation of antibody derived therapeutics and diagnostics. This work presents the optimised production method for HER2-specific sdAb C7b, which led to an ∼11-fold increase in protein yield. In addition, the in vitro and in vivo efficiencies of the targeted delivery of a model nanoparticle cargo (50 nm silica particles doped with Mo6 phosphorescent clusters) conjugated to C7b against those conjugated to HER2-specific trastuzumab is benchmarked. Specifically, this paper demonstrates the significantly higher rate of accumulation in and excretion from xenograft cancer tissue of nanoparticles with C7b, which is of particular importance for diagnostics, i.e. delivery of imaging agents.

Bovine serum albumin encapsulation of near infrared fluorescent nano-probe with low nonspecificity and cytotoxicity for imaging of HER2-positive breast cancer cells

Breast cancer with HER2 overexpressing type links to malignant tumor growth and poor clinical outcome. Successful development of sensitive and selective nano-probe for identification of HER2-positive breast cancer cells is of great importance for breast cancer early diagnosis, subtype classification, and treatment planning. Herein, we report a HER2 antibody conjugated near infrared (NIR) emitted MnCuInS/ZnS qumtun dots (QDs) encapsulated bovine serum albumin (BSA) nano-probe for accurately targeted imaging of HER2-positive breast cancer cells. This NIR nano-probe shows good biocompatibility, low nonspecificity and cytotoxicity, high colloidal stability, and allows HER2-positive breast cancer cell identification with good selectivity. The practicality of this targeted NIR fluorescent nano-probe was proved by successful identifying HER2-positive breast cancer cells from HER2-negative breast cancer cells, which indicates that it can be efficiently applied in selective screening of HER2 overexpressing cancer cells, and provide a platform for the strategy design on the distinction of different breast cancer subtypes.

Immunological Responses against HER2-targeted Idarubicin-ZHER2 Conjugate in BALB/c Mice.

Targeting of cancerous cells with a high level of human epidermal growth factor receptor 2 (HER2) expressions by drug immunoconjugates is a new approach for specific delivery of chemotherapeutic agents. Our previous work indicated that idarubicin-ZHER2 affibody conjugate has a great potential for the treatment of HER2-overexpressing malignant cell lines but possible induced immune response against constructed conjugate was not addressed. In the current study, the possibility of induction of humoral and cellular immune responses against idarubicin-ZHER2 affibody conjugate in BALB/c mice was investigated. For assessment of the induced immune response, prepared and qualified idarubicin-ZHER2 affibody conjugate was administrated intravenously to BALB/c mice and the induced cellular immune response was evaluated by measuring secretion levels of interferon gamma (IFN-γ) and interleukin 10 (IL-10) cytokines by the splenocytes. Humoral response of treated mice was also assessed by measuring total immunoglobulin G (IgG) titer in mice sera. The obtained results showed that idarubicin-ZHER2 affibody conjugate at any examined concentrations could not induce secretion of IFN-γ as a pro-inflammatory cytokine. A mild increase in the level of regulatory IL-10 cytokine was seen in the treated mice although no dose dependency in the level of IL-10 production was observed. Furthermore, results showed that idarubicin-ZHER2 conjugate could not induce IgG production in the treated mice. Based on these findings, the idarubicin-ZHER2 conjugate can be considered as a candidate for the development of new therapeutics against HER2-overexpressing cancers although further in vivo studies are needed.

Near-Infrared Photoimmunotherapy Using a Small Protein Mimetic for HER2-Overexpressing Breast Cancer.

Near-infrared photoimmunotherapy (NIR-PIT) is a new and promising cancer therapy based on a monoclonal antibody conjugated to a photosensitizer which is activated by near-infrared light irradiation, causing cell death. We investigated NIR-PIT using a small protein mimetic (6–7 kDa), Affibody molecules, instead of a monoclonal antibody for HER2-overexpressing cancer. Because of its small size, the Affibody has rapid clearance, high imaging contrast, and good tumor penetration. Due to the small size of the Affibodies, which can cross the blood–brain barrier, NIR-PIT using Affibodies has the potential to extend the target cancer of NIR-PIT, including brain metastases. In vitro, NIR-PIT using HER2 Affibody–IR700Dye conjugates induced the selective destruction of HER2-overexpressing breast cancer cells without damage to control cells having low level expression of HER2. HER2-overexpressing cancer cells showed necrotic cell death and their viability maintained at low levels, even 5 days after NIR-PIT. In contrast, treatment with high concentration of HER2 Affibody–IR700Dye conjugate alone or irradiation with high dose of NIR light alone was without effect on cell viability. Affibody and IR700Dye are currently used clinically, and therefore, we would expect the current formulation to be safely and quickly transitioned into clinical trials.

Comparative anti-proliferative effects of potential HER2 inhibitors on a panel of breast cancer cell lines

Breast cancer is one of the most lethal types of cancer in women worldwide. The human epidermal growth factor receptor 2 (HER2) is considered as a validated target in breast cancer therapy. Previously, we have used quantitative structure activity relationship QSAR equations and their associated pharmacophore models to screen for new promising HER2 structurally diverse inhibitory leads which were tested against HER2-overexpressing SKOV3 ovarian cancer cell line. In this study, we sought to explore the effect of most active ligands against different normal and breast cancer cell lines that represent different breast cancer subtypes with distinguished expression levels in HER2 and HER1. We have tested the promising compounds against SKBR3, MDA-MB-231, MCF7, human fibroblast, and MCF10 cell lines. To understand the inhibitory effects of the active ligands against HER2 over expressed breast cancer cell lines, all inhibitors and the control compound, lapatinib, were docked into the active site of HER2 enzyme performed using Ligand Fit docking engine and PMF scoring function. Five ligands exhibited promising results with relatively low IC50 values on cells that amplify HER2 and high IC50 on those that do not express such a receptor. The most potent compound (compound 13) showed an IC50 of 0.046µM. To test their toxicity against normal cells, the active compounds were tested against both normal fibroblast and normal breast cancer cell MCF-10 and relatively high IC50 values were scored. The IC50 values on HER2 over-expressed breast cancer and normal fibroblast cells provided a promising safety index. Docking results showed the highest similarity in the binding site between the most active ligand and the lapatinib. Our pharmacophore model resulted in a high potent ligand that shows high potency against HER2 positive breast cancer and relatively low toxicity towards the normal human cells.

HER2 Regulates Cancer Stem Cell Activities via the Wnt Signaling Pathway in Gastric Cancer Cells

Introduction: Human epidermal growth factor 2 (HER2) gene overexpression in breast carcinoma cell lines has been shown to drive mammary carcinogenesis and tumor growth and invasion through its effects on mammary stem cells. Objective: Therefore, we investigated the mechanism by which HER2 regulates cancer stem cell (CSC) activity in gastric cancer cells. Methods: HER2 was transfected into MKN28 gastric cancer cells, and its role in regulating CSC activity was determined by characterizing the HER2-overexpressing cells. Results: The sphere formation assay revealed that the sphere sizes and frequency of sphere formation were significantly greater for the HER2-overexpressing cells than for the MKN28 control cells. The CSC markers Oct-4 and BMI1 were more highly expressed in the HER2-overexpressing cells, as were the EMT markers. This was accompanied by a significant enhancement in cellular invasion of the Matrigel and migration. The E-cadherin level was significantly downregulated, and the mesenchymal marker Snail upregulated, in the HER2-transfected cells. HER2 overexpression activated the well-characterized CSC-associated Wnt/β-catenin signaling pathway, as shown by the luciferase assay. After treatment of these cells with the Wnt signal inhibitor PRI-724, the BMI1 and Oct-4 levels were decreased for 24 h and Snail was also downregulated. Immunofluorescence staining revealed the significant restoration of E-cadherin levels in the HER2-transfected cells after PRI-724 treatment. Conclusions: These results established a role for HER2 in regulating gastric CSC activity, with Wnt/β-catenin signaling being mediated via a HER2-dependent pathway. In summary, HER2-overexpressing gastric cancer cells exhibited increased stemness and invasiveness and were regulated by Wnt/β-catenin signaling.

Assessment of the Molecular Mechanism of Action of SB3, a Trastuzumab Biosimilar.

BackgroundSB3, a biosimilar of Herceptin® (trastuzumab, hereinafter referred to as reference product) is currently approved in the EU, Korea, Australia, the USA, and Brazil for the treatment of human epidermal growth factor receptor (HER) 2-positive early and metastatic breast cancer and HER2-positive metastatic gastric cancer. Previously, the biological similarity of SB3 to EU- or US-sourced reference product was assessed using various cell-based and binding assays.ObjectiveIn this paper, as a part of its similarity assessment, SB3 was evaluated for additional characteristics related to its molecular mechanism of action (MoA).MethodsFor extracellular effects of SB3, HER2-overexpressing cancer cell lines were used to assess expression of surface HER2, shedding of the extracellular domain of HER2, and antibody-dependent cell-mediated phagocytosis (ADCP) activity. For intracellular effects, Akt phosphorylation and vascular endothelial growth factor (VEGF) release were assessed. Additionally, in vitro docetaxel or pertuzumab combination experiments were performed for further characterization; anti-proliferation, HER2/HER3 dimerization inhibition, apoptosis, and antibody-dependent cell-mediated cytotoxicity (ADCC) assays were used.ResultsIt was confirmed that SB3 is highly similar to the reference products on quality attributes related to extracellular/intracellular efficacy. This similarity was also confirmed during combination studies with docetaxel and pertuzumab.ConclusionOverall, the equivalence of SB3 with reference product in MoA-related qualities in in vitro mono- and combination therapy experiments may support clinical bioequivalence of the two substances.

Detection of circulating tumor cells (CTCs) in cerebrospinal fluid of a patient with HER2-overexpressing gastric cancer and single cell analysis of intra-patient heterogeneity of CTCs.

BackgroundThe purpose of this study was to identify circulating tumor cells (CTCs) through single cell sorting using DEPArray technology in cerebrospinal fluid (CSF) samples from an advanced gastric cancer (AGC) patient with leptomeningeal seeding. We also demonstrated intra-patient heterogeneity of CTCs isolated from CSF samples at the single cell level.MethodsWe used the DEPArray system to identify and align single enabled CSF and CTCs based on a multi-parallel fluorescence analysis. The CSF samples were stained with an antibody cocktail recognizing DAPI, cytokeratin, EpCAM, and HER2. The stained cells were sorted and identified into a single, variable cell based on a multiparametric fluorescence analysis. The CSF and CTC subpopulations were quantified for absolute cell counts and relative frequency. Based on the stained morphology of each single CSF and CTC, five groups were identified.ResultsWe stratified the CTCs to evaluate the character of the CTCs isolated from the CSF, according to CK, EpCAM and HER2. In total, 78.2% of the CTCs isolated from the CSF showed HER2 overexpression, which was consistent with the status of HER2 in the patient’s primary gastric tumor. The most common types were HER2 positive, CK positive, and EpCAM positive CTCs (56.6%). On the other hand, 21.8% of the CTCs isolated from the CSF did not show HER2 overexpression, which was inconsistent with the primary tumor. Her2 negative, CK negative, and EpCAM positive CTCs were the predominant types among the CTCs without HER2 overexpression (19.7%).ConclusionsWe enriched the CTCs through a single cell sorting process with DEPArray technology in CSF samples of an AGC patient with leptomeningeal seeding. We also demonstrated intra-patient heterogeneity of the CTCs at the single cell level.

DARPin_9-29-Targeted Mini Gold Nanorods Specifically Eliminate HER2-Overexpressing Cancer Cells.

We have demonstrated that designed ankyrin repeat protein (DARPin) _9-29, which specifically targets human epidermal growth factor receptor 2 (HER2), binds tightly to gold mini nanorods (GNRs). Molecular dynamic simulations showed that a single layer of DARPin_9-29 molecules is formed on the surface of the nanorod and that conjugation with the nanorod does not involve the protein's domain responsible for specific binding to HER2. The nanorod-DARPin (DARPin-GNR) conjugate is specifically bound (in nanomolar concentrations) to human breast adenocarcinoma SK-BR-3 cells overexpressing HER2. Illumination by near-infrared light (850 nm) led to almost complete eradication of the conjugate-treated SK-BR-3 cells; the viability of epithelial human breast cancer cells expressing normal amounts of the receptor was much less affected by the illumination. The results reported here pave the way toward application of DARPin-GNR conjugates in phototherapy of cancer.

Abstract 355: A novel anti-HER2 antibody that displays superior synergetic effects in Trastuzumab mediated HER2-overexpressing tumor arrest

Abstract The majority of patients with metastatic breast cancer on Trastuzumab, an anti-HER2 monoclonal antibody, generally develop resistance to the drug within a year after initiation of the treatment. Therefore, there is an unmet need for a novel agent to treat HER2 overexpressing breast cancers. Here we described a novel anti-HER2 humanized monoclonal antibody, 19H6-hu, which binds to HER2 ECD with high affinity and could inhibit the proliferation of multiple HER2-overexpressing cancer cell lines as a single agent, though, to a lesser degree when compared to Trastuzumab alone. Interestingly, when in combination with Trastuzumab, 19H6-hu exhibited much stronger potency in inhibition of BT474, N87 and SKBR3 cell growth relative to that of Perjeta in combination with Trastuzumab as well as in suppression of tumor growth in vivo when compared to Trastuzumab alone. Alanine scanning demonstrated that the antibody binds to an alpha-helix located within the domain III and in proximity to the domain IV of HER2 ECD, which is different from the known binding sites of Trastuzumab and Perjeta. Western blotting further confirmed that 19H6-hu in combination with Trastuzumab is more efficacious in blocking phosphorylation of ERK and phosphorylation of HER2 at Y1248 when compared to Trastuzumab in combination with Perjeta or applied alone. Thereby, our results highlight the functional variability of HER2 domains and provide a new insight into the design of HER2-targeting agents. Citation Format: Haomin Huang, xuesai zhang, jianhe Chen, Le Zhao, qingrou Li, zhenping zhu. A novel anti-HER2 antibody that displays superior synergetic effects in Trastuzumab mediated HER2-overexpressing tumor arrest [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 355.

Structural insight into a matured humanized monoclonal antibody HuA21 against HER2-overexpressing cancer cells.

HER2, a member of the epidermal growth factor receptor (EGFR) family, has been associated with human breast, ovarian and gastric cancers. Anti-HER2 monoclonal antibodies (mAbs) have demonstrated clinical efficacy for HER2-overexpressing breast cancer. A chimeric antibody chA21 that specifically inhibits the growth of HER2-overexpressing cancer cells both in vitro and in vivo has previously been developed. To reduce a potential human anti-mouse immune response, the humanized antibody HuA21 was developed and was further subjected to affinity maturation by phage display on the basis of chA21. Here, the crystal structure of HuA21-scFv in complex with the extracellular domain of HER2 is reported, which demonstrates that HuA21 binds almost the same epitope as chA21 and also provides insight into how substitutions in HuA21 improve the binding affinity compared with chA21, which could facilitate structure-based optimization in the future. Furthermore, the effects of HuA21 variants with constant domains of different lengths were explored and it was noticed that the deletion of constant domain 1 could improve the inhibition efficacy in a cell-proliferation assay, possibly functioning via increased internalization, which might guide the design of other monoclonal antibodies.