HER2-expressing Breast Cancer Research Articles

Second line trastuzumab emtansine following horizontal dual blockade in a real-life setting.

Despite relevant medical advancements, metastatic breast cancer remains an uncurable disease. HER2 signaling conditions tumor behavior and treatment strategies of HER2 expressing breast cancer. Cancer treatment guidelines uniformly identify dual blockade with pertuzumab and trastuzumab plus a taxane as best first line and trastuzumab emtansine as preferred second line choice. However, there is no prospectively designed available study focusing on the sequence and outcomes of patients treated with T-DM1 following the triplet. In the following report, data concerning a wide series of patients treated in a real-life setting are presented. Results obtained in terms of response and median progression free survival suggests a significant role for T-DM1 in disease control of metastatic HER2 expressing breast cancer.

Trastuzumab deruxtecan (T-DXd; DS-8201) in combination with pembrolizumab in patients with advanced/metastatic breast or non-small cell lung cancer (NSCLC): A phase Ib, multicenter, study.

TPS1100 Background: T-DXd is an antibody-drug conjugate composed of an anti-HER2 antibody, cleavable tetrapeptide-based linker, and potent topoisomerase I inhibitor. In a phase II trial in patients (pts) with heavily pretreated, metastatic HER2+ breast cancer (BC), T-DXd had a confirmed objective response rate (cORR) of 60.9% (112/184) and median progression-free survival (PFS) of 16.4 mo (Modi N Engl J Med 2019); these results led to the recent FDA approval for HER2+ unresectable or metastatic BC after ≥ 2 prior anti-HER2 based regimens. For HER2-low BC and HER2-expressing/mutated NSCLC, no HER2-directed therapies have been approved. In a phase I trial of T-DXd in pts with HER2-low BC or HER2-expressing/mutated NSCLC, cORR was 44.2% (19/43) (Modi SABCS 2018), and 55.6% (10/18) (Tsurutani Thorac Oncol 2018), respectively. In preclinical models, T-DXd combined with an anti–PD-1 antibody was more effective than monotherapy with either agent (Iwata Mol Cancer Ther 2018). Here we describe a phase Ib study of T-DXd in combination with pembrolizumab in pts with locally advanced/metastatic HER2-expressing BC or HER2-expressing/mutated NSCLC (DS8201-A-U106; NCT04042701). Methods: This is an open-label, multicenter, nonrandomized, multidose, 2-part study in adult (aged ≥18 y) pts in the United States and Europe. In part 1 (dose escalation), pts received T-DXd 3.2 or 5.4 mg/kg IV q3w and pembrolizumab 200 mg IV q3w to determine the recommended dose for expansion (RDE). The RDE will be given to 4 cohorts (part 2): 2 cohorts with BC (HER2+ [IHC 3+ or IHC 2+/ISH+] with progression on prior T-DM1; and HER2-low [IHC 1+ or IHC2+/ISH-] with progression on prior standard treatments) and 2 cohorts with NSCLC (anti–PD-1, –PD-L1, and -HER2 treatment naive either HER2-expressing [IHC ≥ 1+] or HER2-mutated). Enrollment began in February 2020 with approximately 115 to 133 pts planned to be enrolled at 5 sites for part 1 and expanding to 25 sites for part 2. The primary endpoint in part 1 is dose-limiting toxicities. The part 2 primary efficacy endpoint is cORR by independent central review (ICR) per RECIST 1.1. Additional endpoints include duration of response, disease control rate, and progression-free survival by ICR, overall survival, safety, and pharmacokinetics. Clinical trial information: NCT04042701 .

H2Mab-19, an anti-human epidermal growth factor receptor 2 monoclonal antibody exerts antitumor activity in mouse oral cancer xenografts.

Human epidermal growth factor receptor 2 (HER2) is reported to be overexpressed in breast cancers and is associated with poor clinical outcome. Trastuzumab is a humanized anti-HER2 antibody that offers significant survival benefits to patients with HER2-overexpressing breast cancer. In this study, a novel anti-HER2 monoclonal antibody (mAb), H2Mab-19 (IgG2b, kappa) was developed. Antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antitumor activity of H2Mab-19 were investigated using both breast cancer and oral cancer cell lines. H2Mab-19 demonstrated cytotoxicity in BT-474 (a human breast cancer cell line) and HSC-2 or SAS (human oral cancer cell lines). H2Mab-19 also possessed both ADCC and CDC activity against BT-474, HSC-2, and SAS cell lines. In comparison to control mouse IgG, H2Mab-19 significantly reduced tumor development in BT-474, HSC-2, and SAS xenografts. Collectively, these results suggest that treatment with H2Mab-19 may be a useful therapy for patients with HER2-expressing breast and oral cancers.

SH3BGRL confers innate drug resistance in breast cancer by stabilizing HER2 activation on cell membrane

BackgroundHER2-positive breast cancer is usually associated to the more aggressive progression and the worse prognosis, but the mechanism underlying the innate resistance to HER2-targeted therapy remains elusive. The scaffold protein SH3-domain-binding glutamic acid-rich protein-like protein (SH3BGRL) is indicated as a tumor suppressor in some cancers, but it is highly expressed in breast cancers. Here we characterized the tumorigenic function of SH3BGRL in HER2-expressing breast cancer cells and the subsequent effect in HER2-targeted therapies.MethodsThe interaction of SH3BGRL to HER2 were characterized with various truncated SH3BGRL mutants by immunoprecipitation and molecule docking simulation. The physiological roles of SH3BGRL interacting with HER2 in tumor progression and therapy implication were characterized by gain and loss of function approaches in vitro and in vivo. Immunohistochemistry was used for detections of SH3BGRL and p-HER2 (Y1196) expressions in xenografted tumors and human breast cancer tissues. Clinical relevance of SH3BGRL expression with HER2 was validated with both breast patient sample and the public data analyses.ResultsOur results demonstrated that SH3BGRL directly binds with HER2 on cell membrane via its motifs α1, α2 helixes and β3 sheet, which postpones HER2 internalization upon EGF stimulation. Consequently, the association between SH3BGRL and HER2 contributed to the prolonged HER2 phosphorylation at specific tyrosine sites, especially at Y1196, and their downstream signaling activation. The relevance between SH3BGRL expression and p-HER2 (Y1196) phosphorylation was validated in both xenografted tumors and the breast cancer patient tissues. Mechanistically, SH3BGRL promoted breast tumor cell proliferation and survival, while reduced the cell sensitivity to anti-tumor drugs, especially to the HER2-targeted drugs. In contrast, Silencing SH3BGRL or inhibiting its downstream signals efficiently induced apoptosis of breast tumor cells with HER2 and SH3BGRL doubly positive expression. Database analysis also highlighted that SH3BGRL is a poor prognostic marker, especially for HER2-positive breast cancers.ConclusionsOur results disclose SH3BGRL as a novel posttranslational modulator of HER2 hyperactivation, which can lead to the intrinsic resistance to HER2-targeted therapy. SH3BGRL would be a pivotal therapy target and a diagnostic marker to HER2-positve patients. Thus, targeting SH3BGRL or the downstream signaling could relieve the innate resistance to some HER2-tageted therapies for both HER2 and SH3BGRL-postive breast cancers.

Prospective, randomized, single-blinded, multi-center phase II trial of two HER2 peptide vaccines, GP2 and AE37, in breast cancer patients to prevent recurrence

PurposeAE37 and GP2 are HER2 derived peptide vaccines. AE37 primarily elicits a CD4+ response while GP2 elicits a CD8+ response against the HER2 antigen. These peptides were tested in a large randomized trial to assess their ability to prevent recurrence in HER2 expressing breast cancer patients. The primary analyses found no difference in 5-year overall disease-free survival (DFS) but possible benefit in subgroups. Here, we present the final landmark analysis.MethodsIn this 4-arm, prospective, randomized, single-blinded, multi-center phase II trial, disease-free node positive and high-risk node negative breast cancer patients enrolled after standard of care therapy. Six monthly inoculations of vaccine (VG) vs. control (CG) were given as the primary vaccine series with 4 boosters at 6-month intervals. Demographic, safety, immunologic, and DFS data were evaluated.Results456 patients were enrolled; 154 patients in the VG and 147 in CG for AE37, 89 patients in the VG and 91 in CG for GP2. The AE37 arm had no difference in DFS as compared to CG, but pre-specified exploratory subgroup analyses showed a trend towards benefit in advanced stage (p = 0.132, HR 0.573 CI 0.275–1.193), HER2 under-expression (p = 0.181, HR 0.756 CI 0.499–1.145), and triple-negative breast cancer (p = 0.266, HR 0.443 CI 0.114–1.717). In patients with both HER2 under-expression and advanced stage, there was significant benefit in the VG (p = 0.039, HR 0.375 CI 0.142–0.988) as compared to CG. The GP2 arm had no significant difference in DFS as compared to CG, but on subgroup analysis, HER2 positive patients had no recurrences with a trend toward improved DFS (p = 0.052) in VG as compared to CG.ConclusionsThis phase II trial reveals that AE37 and GP2 are safe and possibly associated with improved clinical outcomes of DFS in certain subgroups of breast cancer patients. With these findings, further evaluations are warranted of AE37 and GP2 vaccines given in combination and/or separately for specific subsets of breast cancer patients based on their disease biology.

Evaluation of HER2 expression in urothelial carcinoma cells as a biomarker for circulating tumor cells.

Detection of circulating tumor cells (CTC) by techniques based on epithelial cell adhesion molecule (EpCAM) is suboptimal in urothelial carcinoma (UC). As HER2 is thought to be broadly expressed in UC, we explored its utility for CTC detection. HER2 and EpCAM expression was analyzed in 18 UC cell lines (UCCs) by qRT-PCR, western blot and fluorescence-activated cell scanning (FACS) and compared to the strongly HER2-expressing breast cancer cell line SKBR3 and other controls. HER2 expression in UC patient tissues was measured by qRT PCR and correlated with data on survival and risk for metastasis. UCCs with high EpCAM and variable HER2 expression were used for spike-in experiments in the CellSearch system. Twenty-one blood samples from 13 metastatic UC patients were analyzed for HER2-positive CTCs with CellSearch. HER2 mRNA and protein were broadly expressed in UCC, with some heterogeneity, but at least 10-fold lower than in the HER-2+ SKBR3 cells. Variations were unrelated to cellular phenotype or clinicopathological characteristics. EpCAM expression was essentially restricted to UCCs with epitheloid phenotypes. Heterogeneity of EpCAM and HER2 expression was observed also in spike-in experiments. The 7 of 21 blood samples from 6 of 13 patients were enumerated as CTC positive via EpCAM, but only one sample stained weakly positive (1+) for HER2. Detection rate of CTCs by EpCAM in UC is poor, even in metastatic patients. Because of its widespread expression, particularly in patients with high risk of metastasis, detection of HER2 could improve identification of UC CTCs, which is why combined detection using antibodies for EpCAM and HER2 may be beneficial.

Abstract P1-18-12: A phase 1, multicenter, open-label study to assess the effect of [fam-] trastuzumab deruxtecan (T-DXd; DS-8201a) on QTc and pharmacokinetics in subjects with HER2-expressing metastatic and/or unresectable breast cancer

Abstract Background [Fam-] trastuzumab deruxtecan (T-DXd; formerly DS-8201a) is a novel antibody-drug conjugate with a humanized anti-HER2 antibody, peptide-based cleavable linker, and topoisomerase I inhibitor payload (MAAA-1181a). T-DXd has a drug-to-antibody ratio of ≈ 8, and the membrane permeability of the cleaved payload induces a cytotoxic bystander effect. In the phase 1 study (NCT02564900), T-DXd 5.4 and 6.4 mg/kg had a manageable safety profile and confirmed objective response rates (ORRs) of 59.5% in subjects with advanced HER2-positive breast cancer (BC) and 44% in subjects with BC with low HER2 expression. Methods This phase 1 study of T-DXd assessing the QTc interval (by 12-lead ECG) and pharmacokinetics (PK) in subjects with HER2-expressing metastatic BC (NCT03366428) was conducted at 7 study sites in Japan. Subjects received T-DXd 6.4 mg/kg IV infusion once every 3 weeks. Data cutoff (Dec 5, 2018) occurred after all subjects completed ≥ 3 cycles (C) or discontinued. Results 51 subjects enrolled and received T-DXd; all were female and Asian. Median age was 56 y (range, 31-79; ≥ 65 y, 25%); 8% were HER2-positive (IHC3+ or IHC2+/ISH+) and 92% were HER2-low (IHC1+, 73%; IHC 2+/ISH−, 12%); 75% were estrogen-receptor positive. Prior therapies included trastuzumab (24%), CDK4/6i (22%), T-DM1 (16%), and pertuzumab (16%); 67% had received > 5 lines of therapy. At data cutoff, 63% of subjects remained on treatment; primary reasons for discontinuation were disease progression (29%) and adverse events (AEs, 4%; grade [Gr] 2 pneumonitis and Gr 2 LVEF decrease). Median duration of survival follow-up was 4.8 months (range, 1.4-8.9). T-DXd 6.4 mg/kg was not associated with a clinically meaningful QTcF prolongation (change of > 10 ms); the upper bound of the 90% CI was < 10 ms at all assessments. The PK profile of total anti-HER2 antibody was similar to that of T-DXd; MAAA-1181a exposure was low. Some accumulation (35%) of T-DXd was observed from C 1-3 (Table); mean accumulation ratio for AUCtau at C 3 was consistent with the observed t1/2 of T-DXd. All subjects had treatment-emergent AEs (TEAEs; 67% Gr ≥ 3), most of which were gastrointestinal or hematologic disorders and primarily Gr 1 or 2. Most common Gr ≥ 3 TEAEs were neutrophil count decreased (45%), white blood cell count decreased (24%), anemia (10%), platelet count decreased (8%), lymphocyte count decreased (8%), and fatigue (6%). 2 subjects had serious TEAEs (Gr 2 nausea [drug related] and Gr 3 femur fracture [not drug related]). 1 subject had ILD (Gr 2 pneumonitis) and 4 had Gr 1 electrocardiogram QT prolonged. No grade 5 events occurred. The unconfirmed ORR (CR + PR) was 39% (95% CI, 26%-54%), with responses seen in HR+ (37%), HR− (46%), and HER2-low (IHC 1+, 43%; IHC 2+/ISH−, 20%) BC. Median time to response was 2.9 months (range, 1.2-4.4), and the duration of response was not yet reached. The disease control rate (CR + PR + SD ≥ 5 weeks from first dosing date) was 84% (95% CI, 82%-99%). Conclusion T-DXd 6.4 mg/kg was not associated with clinically relevant QTcF prolongation; the accumulation of T-DXd from C 1-3 was consistent with the elimination t1/2. T-DXd had a manageable safety profile, consistent with other studies. T-DXd showed preliminary antitumor activity with clinically meaningful responses in heavily pretreated subjects with metastatic HER2-expressing BC (mostly HER2-low), including HR+ and HR− disease. Pharmacokinetic Parameters for 3 Analytes by CycleMean (SD)Median (range)Cmax, μg/mLAUCtau, μg·d/mLt1/2, dTmax, hT-DXdCycle 1 (n = 51)179 (112)677 (141)5.82 (1.11)2.08 (1.55-7.02)Cycle 3 (n = 37)154 (23.3)905 (189)7.40 (1.48)2.12 (0.70-7.15)AR Cycle 3/ Cycle 1-1.35 (0.150)--Total Anti-HER2 AntibodyCycle 1 (n = 51)165 (110)752 (185)6.35 (2.01)2.07 (1.48-7.02)Cycle 3 (n = 37)142 (27.0)1030 (256)8.27 (1.97)2.07 (0.60-7.15)AR Cycle 3/ Cycle 1-1.36 (0.219)--MAAA-1181aCmax, ng/mLAUCtau, ng·d/mLt1/2, dTmax, hCycle 1 (n = 51)12.6 (4.49)39.0 (11.2)5.74 (1.29)6.93 (3.88-191.47)Cycle 3 (n = 37)9.60 (3.89)41.5 (13.8)6.57 (1.81)6.92 (1.95-70.65)AR Cycle 3/ Cycle 1-1.09 (0.194)--AR, accumulation ratio; AUCtau, area under plasma concentration-time curve over dosing interval; Cmax, maximum concentration; HER2, human epidermal growth factor receptor 2; t1/2, half-life; Tmax, time to maximum concentration. Citation Format: Toshinari Yamashita, Akihiko Shimomura, Toshimi Takano, Yasuaki Sagara, Junichiro Watanabe, Eriko Tokunaga, Kunika Kikumori, Emi Kamiyama, Takahiro Kamio, Ryo Nakamura, Tetsu Shinkai, Shunji Takahashi. A phase 1, multicenter, open-label study to assess the effect of [fam-] trastuzumab deruxtecan (T-DXd; DS-8201a) on QTc and pharmacokinetics in subjects with HER2-expressing metastatic and/or unresectable breast cancer [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 P1-18-12.

The Influence of Glycans-Specific Bioconjugation on the FcγRI Binding and In vivo Performance of 89Zr-DFO-Pertuzumab.

Rationale: The overwhelming majority of radioimmunoconjugates are produced via random conjugation methods predicated on attaching bifunctional chelators to the lysines of antibodies. However, this approach inevitably produces poorly defined and heterogeneous immunoconjugates because antibodies have several lysines distributed throughout their structure. To circumvent this issue, we have previously developed a chemoenzymatic bioconjugation strategy that site-specifically appends cargoes to the biantennary heavy chain glycans attached to CH2 domains of the immunoglobulin's Fc region. In the study at hand, we explore the effects of this approach to site-specific bioconjugation on the Fc receptor binding and in vivo behavior of radioimmunoconjugates.Methods: We synthesized three desferrioxamine (DFO)-labeled immunoconjugates based on the HER2-targeting antibody pertuzumab: one using random bioconjugation methods (DFO-nsspertuzumab) and two using variants of our chemoenzymatic protocol (DFO-sspertuzumab-EndoS and DFO-sspertuzumab-βGal). Subsequently, we characterized these constructs and evaluated their ability to bind HER2, human FcγRI (huFcγRI), and mouse FcγRI (muFcγRI). After radiolabeling the immunoconjugates with zirconium-89, we conducted PET imaging and biodistribution studies in two different mouse models of HER2-expressing breast cancer.Results: MALDI-ToF and SDS-PAGE analysis confirmed the site-specific nature of the bioconjugation, and flow cytometry and surface plasmon resonance (SPR) revealed that all three immunoconjugates bind HER2 as effectively as native pertuzumab. Critically, however, SPR experiments also illuminated that DFO-sspertuzumab-EndoS possesses an attenuated binding affinity for huFcγRI (17.4 ± 0.3 nM) compared to native pertuzumab (4.7 ± 0.2 nM), DFO-nsspertuzumab (4.1 ± 0.1 nM), and DFO-sspertuzumab-βGal (4.7 ± 0.2 nM). ImmunoPET and biodistribution experiments in athymic nude mice bearing HER2-expressing BT474 human breast cancer xenografts yielded no significant differences in the in vivo behavior of the radioimmunoconjugates. Yet experiments in tumor-bearing humanized NSG mice revealed that 89Zr-DFO-sspertuzumab-EndoS produces higher activity concentrations in the tumor (111.8 ± 39.9 %ID/g) and lower activity concentrations in the liver and spleen (4.7 ± 0.8 %ID/g and 13.1 ± 4.0 %ID/g, respectively) than its non-site-specifically labeled cousin, a phenomenon we believe stems from the altered binding of the former to huFcγRI.Conclusion: These data underscore that this approach to site-specific bioconjugation not only produces more homogeneous and well-defined radioimmunoconjugates than traditional methods but may also improve their in vivo performance in mouse models by reducing binding to FcγRI.

Genetically Engineered Cell-Derived Nanoparticles for Targeted Breast Cancer Immunotherapy.

Exosomes are nanosized membranous vesicles secreted by a variety of cells. Due to their unique and pharmacologically important properties, cell-derived exosome nanoparticles have drawn significant interest for drug development. By genetically modifying exosomes with two distinct types of surface-displayed monoclonal antibodies, we have developed an exosome platform termed synthetic multivalent antibodies retargeted exosome (SMART-Exo) for controlling cellular immunity. Here, we apply this approach to human epidermal growth factor receptor 2 (HER2)-expressing breast cancer byengineering exosomes through genetic display of both anti-human CD3 and anti-human HER2 antibodies, resulting in SMART-Exos dually targeting Tcell CD3 and breast cancer-associated HER2 receptors. By redirecting and activating cytotoxic Tcells toward attacking HER2-expressing breast cancer cells, the designed SMART-Exos exhibited highly potent and specific anti-tumor activity both invitro and invivo. This work demonstrates preclinical feasibility of utilizing endogenous exosomes for targeted breast cancer immunotherapy and the SMART-Exos as a broadly applicable platform technology for the development of next-generation immuno-nanomedicines.

Multimodal image-guided surgery of HER2-positive breast cancer using [111In]In-DTPA-trastuzumab-IRDye800CW in an orthotopic breast tumor model

BackgroundCombining modalities using dual-labeled antibodies may allow preoperative and intraoperative tumor localization and could be used in image-guided surgery to improve complete tumor resection. Trastuzumab is a monoclonal antibody against the human epidermal growth factor-2 (HER2) receptor and dual-labeled trastuzumab with both a fluorophore (IRDye800CW) and a radioactive label (111In) can be used for multimodal imaging of HER2-positive breast cancer. The aim of this study was to demonstrate the feasibility of HER2-targeted multimodal imaging using [111In]In-DTPA-trastuzumab-IRDye800CW in an orthotopic breast cancer model.MethodsTrastuzumab was conjugated with p-isothiocyanatobenzyl (ITC)-diethylenetriaminepentaacetic acid (DTPA) and IRDye800CW-NHS ester and subsequently labeled with 111In. In a dose escalation study, the biodistribution of 10, 30, and 100 μg [111In]In-DTPA-trastuzumab-IRDye800CW was determined 48 h after injection in BALB/c nude mice with orthotopic high HER2-expressing tumors. Also, a biodistribution study was performed in a low HER2-expressing breast cancer model. In addition, multimodal image-guided surgery was performed in each group. Autoradiography, fluorescence microscopy, and immunohistochemically stained slices of the tumors were compared for co-localization of tumor tissue, HER2 expression, fluorescence, and radiosignal.ResultsBased on the biodistribution data, a 30 μg dose of dual-labeled trastuzumab (tumor-to-blood ratio 13 ± 2) was chosen for all subsequent studies. [111In]In-DTPA-trastuzumab-IRDye800CW specifically accumulated in orthotopic HER2-positive BT474 tumors (101 ± 7 %IA/g), whereas uptake in orthotopic low HER2-expressing MCF7 tumor was significantly lower (1.2 ± 0.2 %IA/g, p = 0.007). BT474 tumors could clearly be visualized with both micro-SPECT/CT, fluorescence imaging and subsequently, image-guided resection was performed. Immunohistochemical analyses of BT474 tumors demonstrated correspondence in fluorescence, radiosignal, and high HER2 expression.ConclusionsDual-labeled trastuzumab showed specific accumulation in orthotopic HER2-positive BT474 breast tumors with micro-SPECT/CT and fluorescence imaging and enabled image-guided tumor resection. In the clinical setting, [111In]In-DTPA-trastuzumab-IRDye800CW could be valuable for preoperative detection of (metastatic) tumors by SPECT/CT imaging, and intraoperative localization by using a gamma probe and fluorescence image-guided surgery to improve radical resection of tumor tissue in patients with HER2-positive tumors.

HO-1 drives autophagy as a mechanism of resistance against HER2-targeted therapies

PurposeTargeted therapies have resulted in major advances in the treatment of HER2-positive breast cancers. Despite this, up to 70% of patients will develop resistance to treatment within 2 years and new strategies for targeting resistant disease are needed.MethodsTo identify potential resistance mechanisms, we used the mouse MMTV-NIC-PTEN+/− spontaneous model of HER2-positive breast cancer and the pan-HER family kinase inhibitor sapatinib. Vehicle and sapatinib-treated tumors were evaluated by immunohistochemistry and proteomic analysis. In vitro studies were carried out to define the role of heme oxygenase 1 (HO-1) and autophagy in resistance to sapatinib and lapatinib, another pan-HER family kinase inhibitor.ResultsTreatment of tumor-bearing MMTV-NIC-PTEN+/− mice with sapatinib resulted in delayed tumor progression and increased survival. However, tumors eventually progressed on treatment. Proteomic analysis identified proteins associated with cellular iron homeostasis as being upregulated in the sapatinib-treated tumors. This included HO-1 whose overexpression was confirmed by immunohistochemistry. Overexpression of HO-1 in HER2-expressing SKBR3 breast cancer cells resulted in reduced sensitivity to both pan-HER family kinase inhibitors sapatinib and lapatinib. This was associated with increased autophagy in the HO-1 over-expressing cells. Furthermore, increased autophagy was also seen in the sapatinib-treated tumors. Treatment with autophagy inhibitors was able to increase the sensitivity of the HO-1 over-expressing cells to both lapatinib and sapatinib.ConclusionTogether these data indicate a role for HO-1-induced autophagy in resistance to pan-HER family kinase inhibitors.

Fam-] trastuzumab deruxtecan (DS-8201a)-induced antitumor immunity is facilitated by the anti-CTLA-4 antibody in a mouse model.

[Fam-] trastuzumab deruxtecan (DS-8201a) is a HER2 (ERBB2)-targeting antibody-drug conjugate, composed of a HER2-targeting antibody and a topoisomerase I inhibitor, exatecan derivative, that has antitumor effects in preclinical xenograft models and clinical trials. Recently, [fam-] trastuzumab deruxtecan was reported to enhance antitumor immunity and was beneficial in combination with an anti–PD-1 antibody in a mouse model. In this study, the antitumor effect of [fam-] trastuzumab deruxtecan in combination with an anti–CTLA-4 antibody was evaluated. [Fam-] trastuzumab deruxtecan monotherapy had antitumor activity in an immunocompetent mouse model with EMT6 human HER2-expressing mouse breast cancer cells (EMT6-hHER2). [Fam-] trastuzumab deruxtecan in combination with the anti–CTLA-4 antibody induced more potent antitumor activity than that by monotherapy with either agent. The combination therapy increased tumor-infiltrating CD4+ and CD8+ T cells in vivo. Mechanistically, cured mice with treatment of [fam-] trastuzumab deruxtecan and an anti–CTLA-4 antibody completely rejected EMT6-mock cells similar to EMT6-hHER2 cells, and splenocytes from the cured mice responded to both EMT6-hHER2 and EMT6-mock cells as measured by interferon-gamma release. Taken together, these results indicate that antitumor immunity is induced by [fam-] trastuzumab deruxtecan and is facilitated in combination with anti–CTLA-4 antibody.

A chemical conjugate between HER2-targeting antibody fragment and Pseudomonas exotoxin A fragment demonstrates cytotoxic effects on HER2-expressing breast cancer cells

Conventionally, immunotoxins have been produced as a single polypeptide from fused genes of an antibody fragment and a toxin. In this study, we adopted a unique approach of chemical conjugation of a toxin protein and an antibody fragment. The two genes were separately expressed in Escherichia coli and purified to high levels of purity. The two purified proteins were conjugated using a chemical linker. The advantage of this approach is its ability to overcome the problem of low recombinant immunotoxin production observed in some immunotoxins. Another advantage is that various combinations of immunotoxins can be prepared with fewer efforts, because the chemical conjugation of components is relatively simpler than the processes involved in cloning, expression, and purification of multiple immunotoxins. As a proof of concept, the scFv of trastuzumab and the PE24 fragment of Pseudomonas exotoxin A were separately produced using E. coli and then chemically crosslinked. The new immunotoxin was tested on four breast cancer cell lines variably expressing HER2. The chemically crosslinked immunotoxin exhibited cytotoxicity in proportion to the expression level of HER2. In conclusion, the present study revealed an alternative method of generating an immunotoxin that could effectively reduce the viability of HER2-expressing breast cancer cells. These results suggest the effectiveness of this method of immunotoxin crosslinking as a suitable alternative for producing immunotoxins. [BMB

Abstract 328: ADAM17-induced activation of HER receptors mediate resistance to trastuzumab in a subset of moderate HER2-expressing breast cancer cells

Abstract Currently anti-HER2 therapies are indicated for HER2-positive breast cancer patients that are IHC3+ or FISH positive. However, there have been studies suggesting that some HER2-negative patients may also respond to these treatments. Previously, our laboratory showed that trastuzumab upregulates ADAM17 and ADAM10 levels; and the resultant ligand release and the activation of HER receptors mediates trastuzumab acquired resistance in HER2-positive breast cancer. The aim of this study is to further understand the mechanisms of response and resistance of HER2 targeting agents such as trastuzumab and neratinib in low and moderate HER2-expressing breast cancer cells. The HER2 expression of a panel of eight breast cell lines was assessed by IHC, FISH, western blot, and qRT-PCR. Using cell viability studies we found that in comparison with high (IHC 3+) and low HER2 expressing (IHC 0 or 1+) breast cancer cells, moderately expressing HER2 (2+) cells showed an intermediate response to trastuzumab and neratinib. Three moderate HER2-expressing breast cancer cell lines (IHC 2+), MDA-MB-361, MDA-MB-453 and HCC1569 were shown to respond to increasing doses of neratinib (with an IC50 less than 30nM). However, increasing doses of trastuzumab (up to 120 μg/ml) was unable to decrease the cell viability of these cells to below 50% of the control. The response to trastuzumab and neratinib correlated with basal ADAM17 but not ADAM10 expression. Trastuzumab treatment led to an upregulation of ADAM17 and the shedding of HER ligands in the media, as well as the phosphorylation of HER members. ADAM17 inhibition, using a specific anti-ADAM17 antibody or knockdown of the protein, decreased activation of HER members as well as downstream markers, correlating with reduced cell viability in moderate HER2-expressing cell lines. In addition, the combination of trastuzumab with neratinib was more effective than either single agent in moderate HER2-expressing cell lines. Thus, ADAM17 may play a key role in the resistance to trastuzumab in moderate HER2-expressing breast cancer cells. Combining trastuzumab with an ADAM17 inhibitor or an irreversible pan-HER inhibitor such as neratinib may be an effective therapeutic strategy for targeting breast cancers with moderate HER2 expressing tumors. Citation Format: Katharina Feldinger, Vasanthy Vigneswara, Gillian Murphy, Anthony Kong. ADAM17-induced activation of HER receptors mediate resistance to trastuzumab in a subset of moderate HER2-expressing breast cancer cells [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 328.

Abstract 1547: DNA-encoded bispecific T-cell engagers and antibodies present long-term antitumor activity

Abstract Specific antibody therapy, including monoclonal antibodies and bispecific T cell engagers (BiTEs), are important new tools for cancer immunotherapy. However, these approaches are slow to develop and may be limited in their production thus restricting the patients who can access these treatments. BiTEs exhibit a particularly short half-life and difficult production. The development of an approach allowing simplified development, delivery and in vivo production would be an important advance. Here we describe development of a designed synthetic DNA plasmid, which we optimized to permit high expression of an anti-HER2 antibody (HER2DMAb) and delivered it into animals through adaptive electroporation. HER2DMAb was efficiently expressed in vitro and in vivo, reaching levels of 50ug/ml in mouse sera. Mechanistically, HER2DMAb blocked HER2 signaling and induced antibody-dependent cytotoxicity. HER2DMAb delayed tumor progression for HER2-expressing ovarian and breast cancer models. We next used the HER2DMAb scFv portion to engineer a DNA-encoded BiTE. This HER2DBiTE was expressed in vivo for approximately 4 months after a single administration. The HER2DBiTE was highly cytolytic and delayed cancer progression in mice. These studies illustrate a novel approach to generate DBiTEs in vivo which represent promising immunotherapies for HER2+ tumors including ovarian and potentially other cancers. Citation Format: Alfredo Perales-Puchalt, Elizabeth K. Duperret, Kar Muthumani, David B. Weiner, Xue Yang, Xizhou Zhu, Krzysztof Wojtak, Edgar Tello-Ruiz, Megan C. Wise. DNA-encoded bispecific T-cell engagers and antibodies present long-term antitumor activity [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 1547.

A new agent in the family of antibody-drug conjugates.

Amplification of the HER2 gene occurs in approximately 15% of breast cancers, defining HER2-positive disease. Patients whose tumour overexpresses the HER2 receptor derive benefit from trastuzumab-based treatments. 1 Hortobagyi GN Trastuzumab in the treatment of breast cancer. N Engl J Med. 2005; 353: 1734-1736 Crossref PubMed Scopus (240) Google Scholar The emergence of antikinase treatments (eg, neratinib and lapatinib) and other antibodies (eg, pertuzumab) that target the HER2 receptor have enhanced this class of drugs and have substantially improved outcomes for patients with HER2-positive breast cancer. Trastuzumab duocarmazine in locally advanced and metastatic solid tumours and HER2-expressing breast cancer: a phase 1 dose-escalation and dose-expansion studyTrastuzumab duocarmazine shows notable clinical activity in heavily pretreated patients with HER2-expressing metastatic cancer, including HER2-positive trastuzumab emtansine-resistant and HER2-low breast cancer, with a manageable safety profile. Further investigation of trastuzumab duocarmazine for HER2-positive breast cancer is ongoing and trials for HER2-low breast cancer and other HER2-expressing cancers are in preparation. Full-Text PDF

Stimulation of natural killer cells with a CD137-specific antibody enhances trastuzumab efficacy in xenotransplant models of breast cancer.

Trastuzumab, a monoclonal antibody targeting human epidermal growth factor receptor 2 (HER2; also known as HER-2/neu), is indicated for the treatment of women with either early stage or metastatic HER2(+) breast cancer. It kills tumor cells by several mechanisms, including antibody-dependent cellular cytotoxicity (ADCC). Strategies that enhance the activity of ADCC effectors, including NK cells, may improve the efficacy of trastuzumab. Here, we have shown that upon encountering trastuzumab-coated, HER2-overexpressing breast cancer cells, human NK cells become activated and express the costimulatory receptor CD137. CD137 activation, which was dependent on NK cell expression of the FcγRIII receptor, occurred both in vitro and in the peripheral blood of women with HER2-expressing breast cancer after trastuzumab treatment. Stimulation of trastuzumab-activated human NK cells with an agonistic mAb specific for CD137 killed breast cancer cells (including an intrinsically trastuzumab-resistant cell line) more efficiently both in vitro and in vivo in xenotransplant models of human breast cancer, including one using a human primary breast tumor. The enhanced cytotoxicity was restricted to antibody-coated tumor cells. This sequential antibody strategy, combining a tumor-targeting antibody with a second antibody that activates the host innate immune system, may improve the therapeutic effects of antibodies against breast cancer and other HER2-expressing tumors.

Neutralization of BCL-2/XL Enhances the Cytotoxicity of T-DM1 In Vivo.

One of the most recent advances in the treatment of HER2+ breast cancer is the development of the antibody-drug conjugate, T-DM1. T-DM1 has proven clinical benefits for patients with advanced and/or metastatic breast cancer who have progressed on prior HER2-targeted therapies. However, T-DM1 resistance ultimately occurs and represents a major obstacle in the effective treatment of this disease. Because anti-apoptotic BCL-2 family proteins can affect the threshold for induction of apoptosis and thus limit the effectiveness of the chemotherapeutic payload, we examined whether inhibition of BCL-2/XL would enhance the efficacy of T-DM1 in five HER2-expressing patient-derived breast cancer xenograft models. Inhibition of BCL-2/XL via navitoclax/ABT-263 significantly enhanced the cytotoxicity of T-DM1 in two of three models derived from advanced and treatment-exposed metastatic breast tumors. No additive effects of combined treatment were observed in the third metastatic tumor model, which was highly sensitive to T-DM1, as well as a primary treatment-exposed tumor, which was refractory to T-DM1. A fifth model, derived from a treatment naïve primary breast tumor, was sensitive to T-DM1 but markedly benefited from combination treatment. Notably, both PDXs that were highly responsive to the combination therapy expressed low HER2 protein levels and lacked ERBB2 amplification, suggesting that BCL-2/XL inhibition can enhance sensitivity of tumors with low HER2 expression. Toxicities associated with combined treatments were significantly ameliorated with intermittent ABT-263 dosing. Taken together, these studies provide evidence that T-DM1 cytotoxicity could be significantly enhanced via BCL-2/XL blockade and support clinical investigation of this combination beyond ERBB2-amplified and/or HER2-overexpressed tumors.

DNA-encoded bispecific T cell engagers and antibodies present long-term antitumor activity.

Specific antibody therapy, including mAbs and bispecific T cell engagers (BiTEs), are important new tools for cancer immunotherapy. However, these approaches are slow to develop and may be limited in their production, thus restricting the patients who can access these treatments. BiTEs exhibit a particularly short half-life and difficult production. The development of an approach allowing simplified development, delivery, and in vivo production would be an important advance. Here we describe the development of a designed synthetic DNA plasmid, which we optimized to permit high expression of an anti-HER2 antibody (HER2dMAb) and delivered it into animals through adaptive electroporation. HER2dMAb was efficiently expressed in vitro and in vivo, reaching levels of 50 μg/ml in mouse sera. Mechanistically, HER2dMAb blocked HER2 signaling and induced antibody-dependent cytotoxicity. HER2dMAb delayed tumor progression for HER2-expressing ovarian and breast cancer models. We next used the HER2dMAb single-chain variable fragment portion to engineer a DNA-encoded BiTE (DBiTE). This HER2DBiTE was expressed in vivo for approximately 4 months after a single administration. The HER2DBiTE was highly cytolytic and delayed cancer progression in mice. These studies illustrate an approach to generate DBiTEs in vivo, which represent promising immunotherapies for HER2+ tumors, including ovarian and potentially other cancers.

AstraZeneca buys into Daiichi ADC

AstraZeneca will pay Daiichi Sankyo $1.35 billion to jointly develop the antibody-drug conjugate trastuzumab deruxtecan. The ADC combines a HER2-targeting antibody with a topoisomerase I inhibitor, with the goal of delivering the powerful chemotherapy directly to cancer cells. The companies plan to file for regulatory approval of the drug, now in studies to treat people with HER2-expressing breast and gastric cancers, later this year. Daiichi, which will produce the drug, stands to rake in another $5.5 billion in sales and other milestone payments.