HER2 Extracellular Domain Research Articles

Molecular Docking-based Virtual Screening, Molecular Dynamics and Atoms in Molecules (AIM) Studies to Identify Potential Inhibitor against the Extracellular Region of Human Epidermal Receptor 2

In this study, molecular docking, quantum mechanics and molecular dynamics methods were used to investigate protein-ligand interactions of the epidermal growth factor receptor 2 (HER2 ). The virtual screening was performed by docking among 2000 chemicals derived from the ZINC library to find specific inhibitors. Trastuzumab is the active site in the HER2 extracellular domain and the target area. The scoring function was used to calculate the binding affinity of the found inhibitors to the active site of the HER2. Among the found inhibitors, six ligands were chosen based on suitable electronic structure and energy. The H-bond interaction energies between six ligands and HER2 were investigated and bond critical points were determined for each bond path between the two corresponding atoms by the quantum theory of atoms in molecules. Root mean square deviation, root mean square fluctuation, the radius of gyration and binding free energy were calculated to check and evaluate the stability and mobility of the simulated system using molecular dynamics simulation.

ELISA assay employing epitope-specific monoclonal antibodies to quantify circulating HER2 with potential application in monitoring cancer\xa0patients undergoing therapy with trastuzumab

Circulating HER2 extracellular domain (HER2 ECD) levels were proposed as a surrogate for HER2 tissue expression to monitor breast cancer patients for early relapse or responses to standard or HER2-targeted therapies, such as the monoclonal antibody (mAb) trastuzumab. Currently, available commercial ELISA assays for HER2 ECD rely on antibodies recognizing undisclosed or unknown epitopes. In this work, two ELISA assays employing MGR2 and MGR3 epitope-specific mAbs for HER2 ECD were developed and validated, showing good assay precision and linearity of the dose-response signal within the dynamic range of 0.19–12.50 ng mL−1 and detection limits of 0.76 and 0.75 ng mL−1 for the MGR2 and MGR3 assays, respectively. The developed assay showed a good agreement with two widely used commercial kits for HER2 ECD quantification in serum samples from breast cancer patients. A complete characterization of mAb-HER2 ECD interaction was performed by means of surface plasmon resonance using trastuzumab as control for both epitope mapping and kinetics analysis. The epitopes recognized by the two mAbs showed no overlap with trastuzumab, which was confirmed by trastuzumab interference analysis in serum samples. The method showed to be a practical approach to determine HER2 ECD with a high degree of sensitivity, reliability and recovery in samples containing mAbs-based therapies.

Plasma HER2ECD a promising test for patient prognosis and prediction of response in HER2 positive breast cancer: results of a randomized study - SAKK 22/99

BackgroundThe HER2 extracellular domain shed in blood (HER2ECD) is reported to rise and fall in parallel with HER2+ breast cancer behavior. In this study, we evaluated the clinical relevance of plasma HER2ECD values in patients with metastatic breast cancer treated in the SAKK22/99 trial comparing trastuzumab monotherapy followed by trastuzumab-chemotherapy combination at progression versus upfront combination therapy.MethodsQuantitative assessment of plasma HER2ECD was performed in 133 patients at baseline; after 2–24 h; at 3 weeks; at first response evaluation (8–9 weeks); and at tumor progression. Associations with tumor characteristics, disease course and trial treatment were evaluated.ResultsBaseline HER2ECD levels were stable within 24 h after the first trastuzumab injection. These plasma values correlated positively with the HER2 gene ratio (rs = 0.39, P < 0.001) and HER2 protein expression levels (rs = 0.36, P < 0.001) but not with ER/PR status of the primary tumor. HER2ECD baseline levels were positively associated with the presence of visceral disease (P = 0.05) and poor patients’ outcome (Cox-regression: P = 0.009). Patients with high baseline levels (> 35 ng/ml) had the worst overall survival (P = 0.03) if treated with upfront combination therapy. Conversely, patients with low HER2ECD baseline values (< 15 ng/ml) had longer time to progression on combined trastuzumab-chemotherapy when first treated with trastuzumab monotherapy (P = 0.02). Monitoring HER2ECD levels during the course of the trial revealed significant time (P = 0.001) and time-treatment arm interactions (P = 0.0007). Under upfront trastuzumab alone, the HER2ECD levels remained stable until just before disease progression. In patients responding to combination treatment HER2ECD levels decreased to > 20%.ConclusionsPlasma HER2ECD levels in patients with metastatic breast cancer reflect HER2 disease status. This robust biomarker might help identifying patients without visceral disease profiting from a sequential treatment’s modality. Monitoring HER2ECD levels during trastuzumab monotherapy could help defining the optimal time to introduce chemotherapy.Trial registrationRegistration Number by ClinicalTrials.gov: NCT00004935, Trial number: SAKK22/99. Registered on 27 January 2003.

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.

An updated evaluation of serum sHER2, CA15.3, and CEA levels as biomarkers for the response of patients with metastatic breast cancer to trastuzumab-based therapies

BackgroundThe transmembrane receptor tyrosine kinase HER2 is overexpressed in approximately 15% of breast tumors and correlates with poor clinical prognosis. Several treatments that target HER2 are approved for treatment of HER2-positive metastatic breast cancer. The serum biomarkers most widely used to monitor anti-HER2 therapies in patients with HER2-positive metastatic breast cancer currently are CA15.3 and CEA. Nevertheless, their clinical utility in patients with breast cancer remains a subject of discussion and controversy; thus, additional markers may prove useful in monitoring the therapeutic responses of these patients. The extracellular domain of HER2 can be shed by proteolytic cleavage into the circulation and this shed form, sHER2, is reported to be augmented during metastasis of HER2-positive breast tumors. Here, we studied the clinical usefulness of sHER2, CA15.3, and CEA for monitoring treatment for breast cancer.MethodsWe measured prospectively pretreatment and post-treatment serum levels (day 1, 30, 60 and 90) of these three biomarkers in 47 HER2-positive, metastatic breast cancer patients treated with trastuzumab in combination with paclitaxel. Evaluation of the disease was performed according to the Response Evaluation Criteria in Solid Tumor (RECIST) at day 90.ResultsPatients with progressive disease at day 90 had smaller relative changes between day 1 and day 30 than those with complete, partial or stable responses at day 90: -9% versus -38% for sHER2 (P = 0.02), +23% versus -17% for CA15.3 (P = 0.005) and +29% versus -26% for CEA (P = 0.02). Patients with progressive disease at day 90 were less likely than the other patients to have a relative decrease of > 20% in their biomarker levels at day 30: 6% vs 33% for sHER2 (P = 0.03), 0% vs 27% for CA15.3 (P = 0.03), 4% vs 29% for CEA (P = 0.04). No patient with progressive disease at day 90 had > 20% reduction of the average combined biomarker levels at day 30 whereas 63% of the other patients had (P = 0.003). Moreover, when we analyzed a > 10% reduction of the average biomarker levels no patient with progressive disease at day 90 had a decrease > 10% at day 30 whereas 78% of other patients had (P<0.001, Se = 100%, Sp = 78%).ConclusionWe show that regular measurement of sHER2, CA15.3, and CEA levels is useful for predicting the therapeutic response and for monitoring HER2-targeted therapy in patients with HER2-positive metastatic breast cancer. The average decrease of the three biomarkers with a threshold of > 10% appears to be the best parameter to distinguish patients who go on to have progressive disease from those who will have a complete, partial or stable response.

Identification of an Activating Mutation in the Extracellular Domain of HER2 Conferring Resistance to Pertuzumab.

BackgroundThe aberrant expression of HER2 is highly associated with tumour occurrence and metastasis, therefore HER2 is extensively targeted for tumour immunotherapy. For example, trastuzumab and pertuzumab are FDA-approved monoclonal antibodies that target HER2-positive tumour cells. Despite their advances in clinical applications, emerging resistance to these two HER2-targeting antibodies has hindered their further application. Somatic mutations in HER2 receptor have been identified as one of the major reasons for resistance to anti-HER2 antibodies.MethodsWe analysed the frequency of somatic mutations in various tumour types based on TCGA and COSMIC databases. Then, the effect of the most frequent mutation (S310F) on the interaction between pertuzumab and HER2 was analysed by molecular modelling analysis. The effect of the S310F mutation was further evaluated through multiple in vitro binding experiments and antitumour activity assays.ResultsWe found through bioinformatics analysis that S310F, an activating mutation in the HER2 extracellular domain, was the most frequent mutation in HER2. The S310F mutation was shown to confer resistance of HER2-positive tumour cells to pertuzumab treatment. With molecular modelling analysis, we confirmed the possibility that the S310F mutation might disrupt the interaction between pertuzumab and HER2 as a result of a significant change in the critical residue S310. Further functional analyses revealed that the S310F mutation completely abolished pertuzumab binding to HER2 receptor and inhibited pertuzumab antitumour efficacy.ConclusionWe demonstrated the loss-of-function mechanism underlying pertuzumab resistance in HER2-positive tumour cells bearing the S310F mutation.

The biological activity of bispecific trastuzumab/pertuzumab plant biosimilars may be drastically boosted by disulfiram increasing formaldehyde accumulation in cancer cells

Studies of breast cancer therapy have examined the improvement of bispecific trastuzumab/pertuzumab antibodies interacting simultaneously with two different epitopes of the human epidermal growth factor receptor 2 (HER2). Here, we describe the creation and production of plant-made bispecific antibodies based on trastuzumab and pertuzumab plant biosimilars (bi-TPB-PPB). Using surface plasmon resonance analysis of bi-TPB-PPB antibodies binding with the HER2 extracellular domain, we showed that the obtained Kd values were within the limits accepted for modified trastuzumab and pertuzumab. Despite the ability of bi-TPB-PPB antibodies to bind to Fcγ receptor IIIa and HER2 oncoprotein on the cell surface, a proliferation inhibition assay did not reveal any effect until α1,3-fucose and β1,2-xylose in the Asn297-linked glycan were removed. Another approach to activating bi-TPB-PPB may be associated with the use of disulfiram (DSF) a known aldehyde dehydrogenase 2 (ALDH2) inhibitor. We found that disulfiram is capable of killing breast cancer cells with simultaneous formaldehyde accumulation. Furthermore, we investigated the capacity of DSF to act as an adjuvant for bi-TPB-PPB antibodies. Although the content of ALDH2 mRNA was decreased after BT-474 cell treatment with antibodies, we only observed cell proliferation inhibiting activity of bi-TPB-PPB in the presence of disulfiram. We concluded that disulfiram can serve as a booster and adjuvant for anticancer immunotherapy.

Syntheses and Functional Studies of Self-Adjuvanting Anti-HER2 Cancer Vaccines.

The 9-mer peptide MFCH401 (N: 165-173: DTILWKDIF), which is located in the extracellular domain of HER2, has been predicted to be a novel epitope. Self-adjuvanting anti-HER2 vaccine constructs were designed and synthesized via covalently attaching MFCH401 or its linear tandem repeats (2×MFCH401, 3×MFCH401) to a lipopeptide Pam3 CSK4 via iterative condensation reaction. The in vivo results showed the Pam3 CSK4 -MFCH401 vaccine construct can induce higher antibody titers of IgG and IgM than those of other conjugates, and the analysis of changes in plasma cytokines level indicate the activation of Th1 cells and NK cells. In addition, the Pam3 CSK4 -MFCH401 vaccine conjugate induced a specific immune response to HER2-overexpressing human BT474 cells. Our data clearly indicated that MFCH401 is a promising epitope; moreover, its linear tandem repeats were unsuitable for anticancer vaccine design when conjugating with Pam3 CSK4 , which provided useful evidence for developing further anti-HER2 cancer vaccines.

The HER2 S310F Mutant Can Form an Active Heterodimer with the EGFR, Which Can Be Inhibited by Cetuximab but Not by Trastuzumab as well as Pertuzumab.

G309 or S310 mutations on the HER2 extracellular domain II induce receptor activation. Clinically, S310F is most frequent among HER2 extracellular domain mutations and patients with the S310F mutation without HER2 amplification responded to trastuzumab with or without the pertuzumab combination. However, the ability of S310F mutant to form homodimers or heterodimers with wild-type HER2 and other HER receptors, or their reactivity to trastuzumab and pertuzumab treatments, has not been reported. We overexpressed S310F as well as G309A, G309E and S310Y HER2 mutants and tested their reactivity to trastuzumab and pertuzumab. All mutants reacted to trastuzumab, but S310F mutant did not react to pertuzumab along with S310Y or G309E mutants. Thereafter, we tested the effects of trastuzumab and pertuzumab on 5637 cell line expressing both wild-type HER2 and S310F mutant. The ligand-independent HER2 homodimerization blocking antibody, trastuzumab, did not inhibit the activation of the HER2 receptor, suggesting that the S310F HER2 mutant did not form homodimers or heterodimers with wild-type HER2. Because 5637 cells overexpressed the EGFR, the effects of cetuximab and gefitinib were determined, and both inhibited the activation of HER2 and significantly reduced cell growth. Because pertuzumab did not inhibit the phosphorylation of HER2 while it bound to wild-type HER2, EGFR-mediated phosphorylation is expected to occur on the S310F mutant. To confirm whether the S310F mutant HER2 retained its affinity to the EGFR, single molecule interaction analyses using TIRF microscopy were performed, which showed that S310F mutant successfully formed complexes with EGFR. In conclusion, HER2 S310F mutant can form an active heterodimer with the EGFR and it can be inhibited by cetuximab, but not by trastuzumab in combination with pertuzumab.

HER2-Specific Reduction-Sensitive Immunopolymersomes with High Loading of Epirubicin for Targeted Treatment of Ovarian Tumor.

Monoclonal antibodies can effectively target to tumors in patients, as validated by antibody-drug conjugates (ADCs). The clinically used ADCs, nevertheless, are restricted to toxins only and suffer from low drug content, excessive use of antibody, and high cost. Here, we report on trastuzumab-decorated disulfide-cross-linked polymersomes (Tra-Ps) for specific delivery of epirubicin hydrochloride (EPI·HCl) to HER2-positive SKOV-3 ovarian tumor. EPI·HCl-loaded Tra-Ps (Tra-Ps-EPI) with a small size of 50-60 nm and varying Tra surface densities (0.5 to 2.4 Tra per Ps) were conveniently obtained via post-conjugation of thiolated trastuzumab onto the surface of maleimide-functionalized Ps-EPI with a drug loading content of 12.7 wt %. Interestingly, Tra-Ps with 1.3 trastuzumab on the surface exhibited a 6-fold higher binding affinity to the HER2 extracellular domain than that of native trastuzumab. In vitro studies revealed that Tra-Ps-EPI with long-term storage stability could rapidly release drugs under a reductive condition and efficiently deliver a large amount of EPI·HCl to HER2-positive SKOV-3 cells, leading to stronger cytotoxicity than the nontargeted Ps-EPI. Moreover, Tra-Ps-EPI displayed a long circulation time (ca. 8 h), deep tumor penetration, and superior tumor growth inhibition in SKOV-3 ovarian tumor-bearing nude mice, which were more effective than free EPI·HCl and nontargeted Ps-EPI. These HER2-specific reduction-sensitive immunopolymersomes with high loading of epirubicin emerge as an attractive treatment for HER2-positive tumors.

HER2 somatic mutation analysis in breast cancer: correlation with clinicopathological features

HER2 mutations have been reported in approximately 2% of breast cancers. Regardless of HER2 overexpression or amplification status, breast cancer with HER2 mutations may respond to HER2-targeted therapy. As HER2 mutation is rare, the clinical and pathological features of HER2-mutated breast cancers, such as hormonal status, histological grade, and metastasis, remain poorly defined. Therefore, the identification of HER2-mutated breast cancer has clinical significance. We retrospectively screened patients with metastatic breast cancer in whom molecular profiling had been performed using next-generation sequencing from 2012 to 2015; we identified 18 patients with HER2 mutation. Mutations were found on next-generation sequencing-based panels, including Ion AmpliSeq Cancer Hotspot, Oncomine, FoundationOne, and Guardant360. HER2 mutations were identified in both the tyrosine kinase (n = 14) and extracellular (n = 4) domains. Of the 14 cases with tyrosine kinase domain mutations, 13 were estrogen receptor positive; the 4 cases with extracellular domain mutations were exclusively estrogen receptor negative. In addition, 11 of 14 patients with tyrosine kinase domain mutations had bone metastasis, whereas no patients with HER2 extracellular domain mutations had bone metastasis. Histologically, 13 patients had invasive ductal carcinoma, 1 had metaplastic carcinoma, and 4 had invasive lobular carcinoma (ILC). All 4 ILCs were high grade and pleomorphic, and not only had an HER2 mutation in the kinase domain but also had an HER2 mutation involving the L755 site. Specific mutation sites may be involved in the pathogenesis of nonclassic ILC.

Abstract CT017: A Phase Ib of a combination of two chimeric (Trastuzumab-like and Pertuzumab-like) HER-2 B cell peptide vaccine emulsified in ISA 720 and nor-MDP adjuvant in patients with advanced solid tumors

Abstract Background: HER-2 is a transmembrane receptor that is overexpressed in multiple epithelial tumors. We have previously shown that a first generation HER-2 B-cell peptide vaccine targeting epitopes (628-647) and (316-339) was safe and relatively active. We recently developed a novel B-cell epitope specific vaccine (HER-Vaxx) consisting of epitopes derived from the extracellular domain of HER-2 that correspond to amino acid sequences 597-626 (pertuzumab binding site) and 266-296 (trastuzumab binding site). Methods: This first-in-human Phase 1b study evaluated the safety, optimal immunologic/biologic dose (OID/OBD) and immunogenicity of HER-Vaxx. Using a 3+3 dose escalation design, 3 patients (pts) were treated at each dose level (DL). If &amp;lt;2 pts experience dose-limiting toxicity (DLT), we required an additional 3 pts to be entered at that DL for OID/OBD assessment (defined as the dose level inducing a strong immunogenic response for 5 out of 6 patients). We also required that at least 6 patients at each DL to receive 3 inoculations of the combination vaccines at 3-week intervals and that &amp;lt; 2 of the 6 pts experience DLT before dose escalation. DLs were 1.0 mg (DL1), 1.5 mg (DL2), 2.0 mg (DL3) and 2.5 mg (DL4) of each peptide. Each dose contained 0.025 mg of n-MDP. Results: 49 patients with metastatic and/or recurrent solid tumors and a median of 4 prior lines of chemotherapy received at least one inoculation of HER-vaxx. Common related toxicities include injection site reactions (56% grade 1/2 and 5% grade 3/4). There were no DLTs. The OID/OBD was found to be DL 2 (1.5 mg of each peptide vaccine).A total of 28 patients received ≥ 3 vaccinations including 2 with a partial response and 14 with stable disease. Of those, 6 patients received at least one 6-month boost. The vaccine generated sustained humoral response eliciting HER-2 specific antibodies in the majority of responding patients. Conclusion: We demonstrate that HER-Vaxx is well tolerated and able to generate sustained anti-HER-2 immune response. Given the promise, continuous development of the vaccine is ongoing at the suggested OBD in HER-2 overexpressing tumors. Citation Format: Tanios Bekaii-Saab, Lai Wei, Robert Wesolowski, Daniel Ahn, Christina Wu, Maryam Lustberg, Amir Mortazavi, Bhuvaneswari Ramaswamy, Jay Overholser, Pravin Kaumaya. A Phase Ib of a combination of two chimeric (Trastuzumab-like and Pertuzumab-like) HER-2 B cell peptide vaccine emulsified in ISA 720 and nor-MDP adjuvant in patients with advanced solid tumors [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 CT017.

Abstract 3617: Trastuzumab-conjugated gold nanoparticles as novel HER2-targeted therapeutics against trastuzumab-resistant gastric cancer

Abstract While trastuzumab (Tmab), a monoclonal antibody that targets the human epidermal growth factor receptor 2 (HER2), has greatly contributed to improve survival outcomes in metastatic gastric cancer, an issue of concern is that no current HER2-targeted therapeutic agent is effective against Tmab-resistant gastric cancer. Nanotechnology, which has progressed rapidly in recent years, greatly contributes to progress in medical fields including cancer therapy, and especially, gold nanoparticles (AuNPs) are attention-grabbing nanomaterials that are promising drug carriers with unique properties of high in vivo stability and a large surface area available for attachment of materials such as antibodies. Methods: We created HER2-targeted AuNPs by conjugating Tmab onto their surface (T-AuNPs) and examined their therapeutic efficacy and cytotoxic mechanisms using HER2-postive Tmab-resistant (MKN7) or Tmab-sensitive (NCI-N87) gastric cancer cell lines in vitro and in vivo. We also examined T-AuNPs on HER2-negative MKN74 gastric cancer cells with adenoviral vector-mediated overexpression of the HER2 extracellular domain (HER2-ECD). Results: The size of T-AuNPs was 85.39 ± 0.68 nm and the surface was negatively charged with 39.43 ± 0.85 mV. In in vitro assays, T-AuNPs showed 6-fold higher cytotoxic activity than Tmab against NCI-N87 cells. Then, T-AuNPs showed significantly stronger cytotoxic effects than controls against both MKN7 and NCI-N87 cells although Tmab had no effect on MKN7 cells. Autophagy was proven to play an important role in T-AuNPs cytotoxic mechanisms. This autophagy was considered to be induced by HER2-dependent internalization of T-AuNPs, some of which were interestingly located in the cytoplasm independent of lysosomal encapsulation. Although T-AuNPs were not cytotoxic towards MKN74 cells, they became cytotoxic following HER2-ECD overexpression. Finally, Intratumoral injection of T-AuNPs showed potent antitumor effects, which were mediated by autophagy, against NCI-N87 and MKN7 subcutaneous tumors in vivo mouse models. Conclusion: HER2-targeted AuNPs with conjugated Tmab can be a promising strategy for the development of novel therapeutic agents to overcome Tmab resistance in gastric cancer. Citation Format: Kento Kumon, Tetsushi Kubota, Shinji Kuroda, Nobuhiko Kanaya, Yoshihiko Kakiuchi, Tomoko Tsumura, Satoru Kikuchi, Shunsuke Kagawa, Hiroshi Tazawa, Toshiyoshi Fujiwara. Trastuzumab-conjugated gold nanoparticles as novel HER2-targeted therapeutics against trastuzumab-resistant gastric cancer [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 3617.

Abstract 1538: Margetuximab mediates greater Fc-dependent anti-tumor activities than trastuzumab or pertuzumab in vitro

Abstract Margetuximab (M) is an investigational Fc-engineered anti-HER2 monoclonal antibody (mAb) with potential for greater immune-mediated anti-tumor activity than trastuzumab (T). M and T bind the same subdomain IV epitope, but M binds with higher affinity to activating Fc receptor, CD16A (FcγRIIIA), and lower affinity to inhibitory Fc receptor, CD32B (FcγRIIB). Pertuzumab (P) binds to a subdomain II epitope and has the same wild type Fc domain as T. We compared in vitro properties of M, T and P and combinations of M+P and T+P to evaluate Fc domain contributions to anti-HER2 mAb mediated activities. By surface plasmon resonance, M, T and P bound to the HER2 extracellular domain with similar high affinity. Binding affinities of M and T were unchanged if P was pre-bound, indicating lack of interaction. M, T and P exhibited comparable binding to HER2-expressing cell lines. Anti-proliferative activities of M and T toward HER23+ cells (N87, SKBR3 or BT474) in the absence or presence of ligands (EGF or HRG1β) were similar, whereas P exhibited weaker activity. In the absence of ligands, M+P or T+P were comparably as active as M or T alone. In presence of ligands, M+P and T+P were generally more active than M or T alone. Antibody-dependent cell-mediated cytotoxicity (ADCC) was evaluated with N87 and SKBR3 target cells (HER23+, expressing a reporter gene to measure viability) and NK effector cells from donors with differing CD16A158 genotypes. M was 7- to 84-fold more potent than T, as well as 69- to 744-fold more potent than P. Greater differences generally were seen with effector NK cells bearing the more common CD16A158 FF and VF genotypes than with the less common, high affinity CD16A158 VV genotype. While addition of P enhanced mean potency of T by 2- to 3-fold, no such effect was seen with M; however, M or M+P was 7- to 25-fold more potent than T+P. Results with JIMT-1 cells (HER22+) were similar, albeit with lower differential than with HER23+ target cells. NK cells were monitored by flow cytometry following incubation of peripheral blood mononuclear cells with HER2+ target cells and mAbs. M induced greater expression of markers of activation (CD137), cytolytic capability (granzyme B, perforin) and proliferation (Ki67) than T. An anti-HER2 mAb with an inactivated Fc domain was ineffective, indicating that induced changes in NK cells were Fc-dependent. In conclusion, M and T bind HER2 with similar affinity and exhibit comparable anti-proliferative activity that is enhanced by addition of P if ligands (EGF or HRG1β) are present. Relative to T, M mediates superior ADCC with effector cells of all CD16A genotypes and promotes greater NK cell activation and expansion. The M+P combination maintains superior ADCC compared to the T+P combination. Citation Format: Liqin Liu, Yinhua Yang, Robert Burns, Jonathan Li, Haiquan Li, Sergey Gorlatov, Paul Moore, Jeffrey Nordstrom. Margetuximab mediates greater Fc-dependent anti-tumor activities than trastuzumab or pertuzumab in vitro [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 1538.

Combined therapy with HER2 and CDK4/6 inhibitors in HER2+ uterine and ovarian carcinomas: Synergistic effect with combined therapy

Objective: HER2 over-expression/amplification occurs in 11%–30% of ovarian (OC )or endometrial cancer (EC) cases. HER2 is an attractive target especially with new potent strategies to inhibit HER2 functions including irreversible tyrosine kinase inhibitors (TKI) like neratinib. Neratinib has many attractive features including being irreversible, pan-HER receptor TKI with high selectivity and affinity toward HER2. Neratinib has shown efficacy against HER2 amplification/over-expression, with HER2 intracellular or extracellular domain mutations, or truncated HER2 (p95HER2). Cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitors are another class of drugs with potential synergy with anti-HER2 therapy. The objective of this preclinical study was to evaluate the efficacy and signaling pathways altered with neratinib combined with the CDK4/6 inhibitor abemaciclib compared to monotherapy in HER2+ OC and EC.

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.

Comparison of in vitro antitumor activity between SB3 (trastuzumab biosimilar, Ontruzant) and Herceptin combined with an antibody for subdomain II of HER2 in HER2-positive cancer cells.

e14001 Background: The human epidermal growth factor receptor (HER) family plays a critical role in proliferation and survival of cancer cells. Trastuzumab is a recombinant humanized monoclonal antibody that blocks signaling pathways of HER2 tyrosine kinase receptor via binding to subdomain IV of HER2 extracellular domain. A subdomain II targeting therapeutic antibody (DII antibody, pertuzumab) binds to a different region on the same protein. A combination of the two different antibodies might provide a more effective antitumor activity without binding interference [1] . In this study, we evaluate in vitro antitumor activity of SB3 used in the same manner as Herceptin, in combination with a DII antibody that complements the mechanism of action of trastuzumab. [1] Nahta et al, [CANCER RESEARCH 64, 2343–2346, April 1, 2004]. Methods: Similarity assessment of in vitro antitumor activity between SB3 and Herceptin was performed on HER2-overexpressing breast and gastric cancer cells through analyzing HER2 dimerization, cell proliferation and survival activities, and antibody-dependent cellular cytotoxicity (ADCC) in the presence of a DII antibody. Combination index was calculated to evaluate the synergistic effect. Results: SB3 and Herceptin in combination with a DII antibody, showed similar in vitro inhibition rate for HER2/HER3 heterodimerization. The HER2-overexpressing breast and gastric cancer cells growth inhibition rate was similar whether SB3 or Herceptin was used in combination with a DII antibody. This effect was also shown in apoptosis activity by measuring caspase 3/7 expression. In terms of biological effect via Fc function, cell killing activity was assessed by ADCC assay in the presence of a DII antibody, and non-synergistic effect was shown with both SB3 and Herceptin. Conclusions: This study has concluded that SB3, as a trastuzumab biosimilar, demonstrates highly similar in vitro antitumor activity in experimental conditions when combined with a DII antibody, resulting in enhanced cell growth inhibition and apoptosis activities by inhibiting HER2 dimerization in HER2-positive cancer cells.

Multiplexed (18-Plex) Measurement of Signaling Targets and Cytotoxic T Cells in Trastuzumab-Treated Patients using Imaging Mass Cytometry.

Imaging mass cytometry (IMC) uses metal-conjugated antibodies to provide multidimensional, objective measurement of protein targets. We used this high-throughput platform to perform an 18-plex assessment of HER2 ICD/ECD, cytotoxic T-cell infiltration and other structural and signaling proteins in a cohort of patients treated with trastuzumab to discover associations with trastuzumab benefit. An antibody panel for detection of 18 targets (pan-cytokeratin, HER2 ICD, HER2 ECD, CD8, vimentin, cytokeratin 7, β-catenin, HER3, MET, EGFR, ERK 1-2, MEK 1-2, PTEN, PI3K p110 α, Akt, mTOR, Ki67, and Histone H3) was used with a selection of trastuzumab-treated patients from the Hellenic Cooperative Oncology Group 10/05 trial (n = 180), and identified a case-control series. Patients that recurred after adjuvant treatment with trastuzumab trended toward a decreased fraction of HER2 ECD pixels over threshold compared with cases without recurrence (P = 0.057). After exclusion of the lowest HER2 expressers, 5-year recurrence events were associated with reduced total extracellular domain (ECD)/intracellular domain (ICD) ratio intensity in tumor (P = 0.044). These observations are consistent with our previous work using quantitative immunofluorescence, but represent the proof on identical cell content. We also describe the association of the ECD of HER2 with CD8 T-cell infiltration on the same slide. The proximity of CD8 cells as a function of the expression of the ECD of HER2 provides further evidence for the role of the immune system in the mechanism of action of trastuzumab.

In Silico Designing a Candidate Vaccine Against Breast Cancer

Breast cancer (BC) is the most common type of women’s cancer with a prevalence of about 25%, although it is rare in men (< 1%). Until now, BC therapy approaches have not been successful for various reasons. The purpose of this study was to design a candidate vaccine against BC using bioinformatics tools. Herein, in silico design of a fusion vaccine candidate containing extracellular domain of HER-2/neu protein and N-terminal sequence of GP96 protein (NTGP96) were performed. Different bioinformatics and immunoinformatics softwares were used to study the important characteristics of the designed vaccine, including physicochemical properties, secondary, and 3D structures, as well as antigenicity and allergenicity. Structural and immunological computational results indicated the potential of our designed construct for proper stimulation of cellular and humoral immune responses against BC. According to the obtained data, the candidate vaccine could be introduced for BC therapy, after the efficacy of it was confirmed by in vivo and in vitro immunological assays.

Shed HER2 surrogacy evaluation in primary breast cancer patients: a study assessing tumor tissue HER2 expression at both extracellular and intracellular levels

The aim of the present study was to investigate serum HER2 extracellular domain (ECD) as a putative surrogate marker of the shedding phenomenon of HER2 receptor from the tumor tissue of primary breast cancer (BC) patients. A pilot retrospective study was conducted on 100 matched serum and tissue samples from patients with node-positive primary BC, stage II/III. Analysis of association and concordance between serum HER2 ECD levels (measured by chemiluminescence immunoassay) and the expression in matched tumor tissue of HER2 ECD and intracellular receptor domain (ICD) (determined by immunohistochemistry) were performed. The median serum HER2 ECD level was 9.4 ng/ml and cutoff values were set at 15.2 ng/ml or 13.0 ng/ml. HER2 ICD and ECD were overexpressed in tumor tissue of 19.8% and 6.9% of patients, respectively. Statistically significant associations were found between serum HER2 ECD levels and tissue expression of both HER2 ICD and ECD (p < .001; Fisher analysis). Moreover, strong concordances were found between serum HER2 ECD levels and tissue expression of HER2 ICD or ECD (cutoff 15.2 ng/ml: 80 and 92.5%, respectively). Our findings support a role for serum HER2 ECD as a surrogate marker of tissue HER2 status in primary BC, both for HER2 ICD or ECD expression.