anti-HER2 Monoclonal Antibody Research Articles

15P The analgesic compound palmitoylethanolamide reduces inflammation in human cardiomyocytes and vascular endothelial cells exposed to doxorubicin and anti-HER2 monoclonal antibody through PPAR-α and NLRP3-related pathways

Palmitoylethanolamide (PEA) is an endogenous fatty acid mediator that is synthetized from membrane phospholipids by N-acyl phosphatidylethanolamine phospholipase D. It is a new analgesic drug with anti-inflammatory effects through the induction of PPAR-related pathways. We aimed to assess whether palmitoylethanolamide co-incubated during doxorubicin and trastuzumab, reduces anticancer drugs-related cardiotoxicity in cellular models.

1190TiP Pharmacokinetics, efficacy, and safety of patritumab deruxtecan (HER3-DXd) in EGFR inhibitor-resistant, EGFR-mutated (EGFRm) non-small cell lung cancer (NSCLC)

Patients (pts) with advanced EGFRm NSCLC have limited treatment options after progression with an EGFR tyrosine kinase inhibitor (TKI) and platinum-based chemotherapy (PBC). HER3, a member of the human epidermal growth factor receptor family, is expressed across a variety of solid tumors and in most EGFRm NSCLC. HER3 overexpression has been associated with poor clinical outcomes, including metastatic progression and decreased relapse-free survival. HER3-DXd is a novel antibody-drug conjugate composed of a human anti-HER3 monoclonal antibody (patritumab) linked to a topoisomerase I inhibitor payload via a stable tetrapeptide-based, tumor-selective, cleavable linker.

1195TiP HERTHENA-Lung02: A randomized phase III study of patritumab deruxtecan vs platinum-based chemotherapy in locally advanced or metastatic EGFR-mutated NSCLC after progression with a third-generation EGFR TKI

Standard therapies for patients (pts) with epidermal growth factor receptor–mutated (EGFRm) non-small cell lung cancer (NSCLC) that has progressed after treatment with third-generation (3rd-gen) EGFR tyrosine kinase inhibitors (TKIs) offer only a limited benefit. Human epidermal growth factor receptor 3 (HER3) is often expressed in primary NSCLC tumors, and HER3 expression is commonly observed in patients with EGFR mutations. HER3-DXd is a novel antibody-drug conjugate composed of a human anti-HER3 monoclonal antibody (patritumab) linked to a topoisomerase I inhibitor payload via a tetrapeptide-based cleavable linker.

Margetuximab with retifanlimab as first-line therapy in HER2+/PD-L1+ unresectable or metastatic gastroesophageal adenocarcinoma: MAHOGANY cohort A

BackgroundHuman epidermal growth factor receptor 2 (HER2)-positive metastatic gastric and gastroesophageal adenocarcinoma (GEA) is globally treated with chemotherapy plus trastuzumab. Novel therapeutic strategies strive to not only optimize efficacy, but also limit toxicities. In MAHOGANY cohort A, margetuximab, an Fc-engineered, anti-HER2 monoclonal antibody (mAb) was combined with retifanlimab, an anti-programmed cell death protein 1 mAb, in the first-line HER2-positive/programmed death-ligand 1 (PD-L1)-positive GEA.Patients and methodsMAHOGANY cohort A part 1 is a single-arm trial to evaluate margetuximab plus retifanlimab in patients with HER2 immunohistochemistry 3+, PD-L1-positive (combined positive score ≥1%), and non-microsatellite instability-high tumors. Primary objectives for cohort A were safety/tolerability and the confirmed objective response rate (ORR).ResultsAs of 3 August 2021, 43 patients were enrolled and received margetuximab/retifanlimab. Nine grade 3 treatment-related adverse events (TRAEs) were reported in eight (18.6%) patients and eight serious TRAEs in seven (16.3%) patients. There were no grade 4/5 TRAEs. Three patients discontinued margetuximab/retifanlimab because of immune-related adverse events. The ORR by independent assessment was 53% [21/40 (95% confidence interval (CI) 36.1-68.5)], with a median duration of response of 10.3 months (95% CI 4.6-not evaluable); disease control rate was 73% [29/40 (95% CI 56.1-85.4)]. The study sponsor discontinued the study in advance of the planned enrollment when it became apparent that the study design would no longer meet the requirements for drug approval because of recent advances in the treatment of GEA.ConclusionsThe chemotherapy-free regimen of combined margetuximab/retifanlimab as first-line treatment in double biomarker-selected patients demonstrated a favorable toxicity profile compared with historical outcomes using chemotherapy plus trastuzumab. The ORR observed in this study compares favorably versus ORR observed with other chemotherapy-free approaches.

HER2-Displaying M13 Bacteriophages induce Therapeutic Immunity against Breast Cancer.

Simple SummaryThe high incidence and death rates of breast cancer make the development of new therapies an urgent need. The introduction into the clinic of the anti-HER2 monoclonal antibody trastuzumab considerably improved the overall survival and time-to-disease progression of patients with HER2-positive breast cancer. However, many patients do not benefit from it because of resistance to therapy. Cancer vaccines, by inducing into the patient an anti-cancer specific immunity, might represent an alternative immunotherapeutic approach, but despite promises, so far no anti-HER2 cancer vaccine has been approved for human use. In this study, we propose therapeutic phage-based vaccines, against HER2 and its aggressive isoform Δ16HER2, able to elicit a protective immunity and potentially capable of preventing relapse in HER2-positive breast cancer patients, even in those who develop trastuzumab resistance.The advent of trastuzumab has significantly improved the prognosis of HER2-positive (HER2+) breast cancer patients; nevertheless, drug resistance limits its clinical benefit. Anti-HER2 active immunotherapy represents an attractive alternative strategy, but effective immunization needs to overcome the patient’s immune tolerance against the self-HER2. Phage display technology, taking advantage of phage intrinsic immunogenicity, permits one to generate effective cancer vaccines able to break immune tolerance to self-antigens. In this study, we demonstrate that both preventive and therapeutic vaccination with M13 bacteriophages, displaying the extracellular (EC) and transmembrane (TM) domains of human HER2 or its Δ16HER2 splice variant on their surface (ECTM and Δ16ECTM phages), delayed mammary tumor onset and reduced tumor growth rate and multiplicity in ∆16HER2 transgenic mice, which are tolerant to human ∆16HER2. This antitumor protection correlated with anti-HER2 antibody production. The molecular mechanisms underlying the anticancer effect of vaccine-elicited anti-HER2 antibodies were analyzed in vitro against BT-474 human breast cancer cells, sensitive or resistant to trastuzumab. Immunoglobulins (IgG) purified from immune sera reduced cell viability mainly by impairing ERK phosphorylation and reactivating retinoblastoma protein function in both trastuzumab-sensitive and -resistant BT-474 cells. In conclusion, we demonstrated that phage-based HER2 vaccines impair mammary cancer onset and progression, opening new perspectives for HER2+ breast cancer treatment.

Cardiac Safety in Breast Cancer Patients Receiving Pegylated Liposome Doxorubicin Sequential Anti-HER2 Monoclonal Antibody Therapy.

Background: Cardiotoxicity associated with the sequential use of anthracyclines followed by trastuzumab is common in adjuvant therapy of patients with HER2-positive early breast cancer (eBC). However, the cardiac safety of trastuzumab concurrent with pegylated liposomal doxorubicin (PLD) is relatively less studied. Method: Clinical data of patients with HER2-positive eBC treated with PLD and cyclophosphamide (PLD-C) followed by taxanes plus trastuzumab ± pertuzumab (TH or TPH) who then completed standard anti-HER2 treatment for 12 months from June 2012 to August 2021 were retrospectively collected. The primary endpoints were clinical and subclinical cardiotoxicity. Result: In total, 70 eligible patients were enrolled. Among them, 55 patients (78.6%) received PLD-C → TH and 15 patients (21.4%) received PLD-C → TPH. The median follow-up time was 41.8 months. Until August 2021, only two patients had recurrent or metastatic diseases, with 2-year and 5-year disease-free survivals of 98.6% and 96.8%, respectively. Clinical cardiotoxicity occurred in six patients (8.6%), and all of them had an absolute decline of ≥16% from baseline left ventricular ejection fraction (LVEF) but not below the lower limit of normal (LLN = 50%). Subclinical cardiotoxicity events occurred in 17 patients (24.3%), and all of them had absolute declines of ≥10% and <16% from baseline LVEF but not below the LLN. No patients were interrupted from treatment, and all patients completed anti-HER2 treatment for 12 months. The sharpest decrease in LVEF was observed at 18 months after the start of PLD treatment. The cumulative incidences of clinical and subclinical cardiotoxicity were 9.8% and 28.3%, respectively. In the univariate analysis, body mass index, age, left chest wall radiotherapy, and ongoing cardiovascular risk factors were not significantly associated with clinical or subclinical cardiotoxicity (p > 0.05). No patients had congestive heart failure or death caused by PLD or anti-HER2 treatment. Conclusion: The sequential use of PLD and trastuzumab showed a lower incidence of clinical cardiotoxicity, presented as asymptomatic decreased LVEF, compared with the results obtained in previous clinical studies using conventional anthracycline, taxanes and trastuzumab. The study regimen demonstrated good cardiac tolerance and is an alternative strategy for cardioprotection in patients with HER2-positive eBC.

AVB-S6-500, a receptor tyrosine kinase AXL inhibitor, improves the therapeutic efficacy of trastuzumab in uterine serous cancers (278)

<b>Objectives:</b> To determine whether inhibition of <i>GAS6</i>/<i>AXL</i> can improve efficacy to the humanized anti-HER2 monoclonal antibody trastuzumab by decreasing uterine serous cancer cell invasion and cell survival <i>in vitro</i> and tumor burden <i>in vivo</i>. <b>Methods:</b> We treated three uterine serous cancer cell lines (ARK1, ARK2, PUC198) that expressed <i>Her2/neu</i> and <i>AXL</i> receptors with AVB500 (Aravive Biologics, Houston, TX) in combination with Trastuzumab. Clonogenic proliferation assays were performed after three days of treatment, and relative cell viability was calculated using GraphPad Prism. Next, using a Matrigel invasion assay, we examined the invasive capacity of the ARK1 and ARK2 cell lines after 24 hours of treatment with vehicle, AVB500 alone, Trastuzumab alone, or combination Trastuzumab and AVB500. We also performed proximity ligation and IF colocalization assays to show that <i>Her2/neu</i> and <i>AXL</i> receptors form a complex that results in the transphosphorylation and activation of the <i>AXL</i>. We performed <i>in vivo</i> studies using nod-SCID mice injected with 1 x 10⁷ ARK1 and ARK2 cells intraperitoneally, followed by treatment with vehicle control, AVB alone, or Trastuzumab alone or in combination with AVB500 for 35 days and 21 days, respectively. <b>Results:</b> In uterine serous (ARK 2 and PUC 198) cells, there was a significant reduction in cell proliferation when treated with combination Trastuzumab and AVB500 compared to the Trastuzumab-alone group (0.155nm vs 0.202nm, p=0.015; 0.290 vs 0.015, p=0.0044) in ARK 2 and PUC 198 cell lines, respectively. We also observed a reduction in the number of invading tumor cells when cells were treated with combination Trastuzumab and AVB500 versus Trastuzumab alone (3.9 vs 17.7 invading tumor cells/hpf, p=0.0001; 3.2 vs 17.8 invading tumor cells/hpf, p=0.0004) in ARK 1 and ARK 2 cell lines, respectively. Furthermore, the proximity ligation and co-localization assays demonstrated the physical closeness of <i>AXL</i> and <i>HER2</i> receptors in ARK2 cells that could account for the co-regulation of these receptors. Finally, we found that mice treated with Trastuzumab plus AVB -500 developed significantly less tumor burden than mice treated with Trastuzumab alone (0.032 g vs 0.083 g, <i>p</i>= 0.024) in the ARK1 xenograft model and (0.142g vs 0.443 g, <i>p</i>= 0.0015) in the ARK2 xenograft model. <b>Conclusions:</b> The <i>GAS6</i>/<i>AXL</i> inhibitor, AVB-500, potentiated the effect of Trastuzumab to decrease uterine serous cancer cell proliferation and invasion <i>in vitro</i> and tumor burden <i>in vivo</i>. Co-localization of <i>AXL</i> and <i>HER2</i> receptors was seen in both uterine serous cancer cell lines. Our data suggest that the addition of AVB-S6-500 increased the therapeutic efficacy of Trastuzumab therapy in uterine serous cancers. Additional therapeutic and mechanistic experiments are ongoing.

In Vivo and In Vitro Efficacy of Enhertu in Uterine Serous Carcinoma (047)

Objectives: Uterine serous carcinoma (USC) is an aggressive subtype of epithelial endometrial cancer, comprising approximately 40% of endometrial cancer-related deaths. HER2 overexpression is detected in about 35% of patients with USC. Enhertu is a novel humanized monoclonal anti-HER2 antibody (trastuzumab) linked to the topoisomerase I inhibitor payload deruxtecan by a cleavable linker. Enhertu is FDA-approved for HER2-expressing metastatic breast and gastric cancers; however, its efficacy has not been tested in endometrial cancer. The objective of this study was to study the in vitro and in vivo efficacy of Enhertu in USC. Methods: HER2 expression was analyzed with flow cytometry in primary USC cell lines: ARK2, ARK4, ARK7, and ARK20. Cell lines were treated with 0.05, 0.010, 0.025, 0.05, 0.2 μg/ml of Enhertu or Control antibody-drug conjugate (Control ADC). The inhibitory concentration 50 (IC50) was assessed by counting the viable cells using a flow cytometer. Apoptosis and necrosis were assessed with Annexin V and propidium iodide (PI) staining. GFP-expressing ARK4 (ARK4-GFP) cells were co-cultured with ARK2 cells for the bystander effect assay. For in vivo experiments, eight million ARK2 cells were injected subcutaneously into SCID mice. Animals were randomized to intravenous injection of PBS (n=5), control ADC (4 mg/kg, n=6) or Enhertu (4 mg/kg, n=5) treatment. Kruskal Wallis, Friedman, and Log Rank tests were used for statistical analysis. Graphical Abstract View Large Image Figure Viewer Download Hi-res image Graphical Abstract View Large Image Figure Viewer Download Hi-res image Conclusions: Enhertu showed significant preclinical efficacy against HER2 overexpressing uterine serous carcinoma cell lines. Further clinical trials in USC are warranted. Objectives: Uterine serous carcinoma (USC) is an aggressive subtype of epithelial endometrial cancer, comprising approximately 40% of endometrial cancer-related deaths. HER2 overexpression is detected in about 35% of patients with USC. Enhertu is a novel humanized monoclonal anti-HER2 antibody (trastuzumab) linked to the topoisomerase I inhibitor payload deruxtecan by a cleavable linker. Enhertu is FDA-approved for HER2-expressing metastatic breast and gastric cancers; however, its efficacy has not been tested in endometrial cancer. The objective of this study was to study the in vitro and in vivo efficacy of Enhertu in USC. Methods: HER2 expression was analyzed with flow cytometry in primary USC cell lines: ARK2, ARK4, ARK7, and ARK20. Cell lines were treated with 0.05, 0.010, 0.025, 0.05, 0.2 μg/ml of Enhertu or Control antibody-drug conjugate (Control ADC). The inhibitory concentration 50 (IC50) was assessed by counting the viable cells using a flow cytometer. Apoptosis and necrosis were assessed with Annexin V and propidium iodide (PI) staining. GFP-expressing ARK4 (ARK4-GFP) cells were co-cultured with ARK2 cells for the bystander effect assay. For in vivo experiments, eight million ARK2 cells were injected subcutaneously into SCID mice. Animals were randomized to intravenous injection of PBS (n=5), control ADC (4 mg/kg, n=6) or Enhertu (4 mg/kg, n=5) treatment. Kruskal Wallis, Friedman, and Log Rank tests were used for statistical analysis. Conclusions: Enhertu showed significant preclinical efficacy against HER2 overexpressing uterine serous carcinoma cell lines. Further clinical trials in USC are warranted.

IFI16-dependent STING signaling is a crucial regulator of anti-HER2 immune response in HER2+ breast cancer

Relapse to anti-HER2 monoclonal antibody (mAb) therapies, such as trastuzumab in HER2+ breast cancer (BC), is associated with residual disease progression due to resistance to therapy. Here, we identify interferon-γ inducible protein 16 (IFI16)-dependent STING signaling as a significant determinant of trastuzumab responses in HER2+ BC. We show that down-regulation of immune-regulated genes (IRG) is specifically associated with poor survival of HER2+, but not other BC subtypes. Among IRG, IFI16 is identified as a direct target of EZH2, the underexpression of which leads to deficient STING activation and downstream CXCL10/11 expression in response to trastuzumab treatment. Dual inhibition of EZH2 and histone deacetylase (HDAC) significantly activates IFI16-dependent immune responses to trastuzumab. Notably, a combination of a novel histone methylation inhibitor with an HDAC inhibitor induces complete tumor eradication and long-term T cell memory in a HER2+ BC mouse model. Our findings demonstrate an epigenetic regulatory mechanism suppressing the expression of the IFI16-CXCL10/11 signaling pathway that provides a survival advantage to HER2+ BC to confer resistance to trastuzumab treatment.

TRAF4 hyperactivates HER2 signaling and contributes to Trastuzumab resistance in HER2-positive breast cancer

The HER2 receptor modulates downstream signaling by forming homodimers and heterodimers with other members of the HER family. For patients with HER2-positive breast cancer, Trastuzumab, an anti-HER2 monoclonal antibody as first-line therapy has shown significant survival benefits. However, the development of acquired resistance to Trastuzumab continues to be a significant obstacle. TNF receptor-associated factor 4 (TRAF4) upregulation was discovered to be associated with a worse clinical outcome. Here we identified TRAF4 overexpression as one of the putative mechanisms for HER2-positive breast cancer cells to maintain HER2 signaling during Trastuzumab treatment, while TRAF4 knockdown reduced HER2 stability and improved Trastuzumab sensitivity. Mechanistically, TRAF4 regulates HER2 level through its impact on SMAD specific E3 ubiquitin protein ligase protein 2 (SMURF2). The development of a membrane-associated protein complex containing HER2, TRAF4, and SMURF2 has been observed. SMURF2 bound to the HER2 cytoplasmic domain, and directly ubiquitinated it leading to HER2 degradation, whereas TRAF4 stabilized HER2 by degrading SMURF2 and inhibiting the binding of SMURF2 to HER2. Moreover, downregulation of TRAF4 has decreased the AKT/mTOR signaling. In conclusion, we discovered a new HER2 signaling regulation that involves the TRAF4-SMURF2 complex, a possible mechanism that might contribute to anti-HER2 resistance, making TRAF4 a viable target for treating HER2 + breast cancer.

Defucosylated mouse‑dog chimeric anti‑HER2 monoclonal antibody exerts antitumor activities in mouse xenograft models of canine tumors.

Human epidermal growth factor receptor 2 (HER2) overexpression has been reported in various types of cancer, including breast, gastric, lung, colorectal and pancreatic cancer. A humanized anti-HER2 monoclonal antibody (mAb), trastuzumab, has been shown to improve survival of patients in HER2-positive breast and gastric cancer. An anti-HER2 mAb, H2Mab-77 (mouse IgG1, kappa) was previously developed. In the present study, a defucosylated version of mouse-dog chimeric anti-HER2 mAb (H77Bf) was generated. H77Bf possesses a high binding-affinity [a dissociation constant (KD): 7.5×10−10 M, as determined by flow cytometric analysis] for dog HER2-overexpressed CHO-K1 (CHO/dHER2) cells. H77Bf highly exerted antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) for CHO/dHER2 cells by canine mononuclear cells and complement, respectively. Moreover, administration of H77Bf significantly suppressed the development of CHO/dHER2 ×enograft tumor in mice compared with the control dog IgG. H77Bf also possesses a high binding-affinity (KD: 7.2×10−10 M) for a canine mammary gland tumor cell line (SNP), and showed high ADCC and CDC activities for SNP cells. Intraperitoneal administration of H77Bf in mouse xenograft models of SNP significantly suppressed the development of SNP xenograft tumors compared with the control dog IgG. These results indicated that H77Bf exerts antitumor activities against dHER2-positive canine cancers, and could be valuable treatment regimen for canine cancers.

Abstract LB088: Targeting ATR enhances the antitumor efficacy of patritumab deruxtecan (HER3-DXd) in tamoxifen-resistant ER+ breast cancer cells by reprogramming cell cycle progression

Abstract Background: HER3, a member of the ErbB family of receptor tyrosine kinases that activate multiple oncogenic signaling pathways, is overexpressed in 50-70% of breast cancers (BC). HER3 mRNA levels are higher in ER+ breast tumors than in other molecular subtypes. Inhibition of ER activity using an antagonist increased HER3 protein expression and activation, which was essential for growth and survival of ER+ BC cells resistant to the ER antagonist. Therefore, therapeutically targeting HER3 with HER3-DXd, an antibody-drug conjugate (ADC) composed of a fully human anti-HER3 monoclonal antibody (patritumab) conjugated to a topoisomerase I inhibitor payload (an exatecan derivative) via a tetrapeptide-based cleavable linker, can be an effective treatment for tamoxifen-resistant (TMR) ER+ BC. After first assessing HER3-DXd as a single agent, we sought to identify a synergistic partner to maximize HER3-DXd’s antitumor activity in HER3+/ER+ TMR BC. Methods: Whole-genome high-throughput siRNA screening was performed to identify targets whose inhibition enhances HER3-DXd’s antitumor efficacy. The antitumor effects of HER3-DXd plus the synergistic partners were assessed using a soft agar colony formation assay and a clonogenic assay in TMR HER3+/ER+ MCF7 and T47D BC cells. Treatment effects on cell cycle distribution, apoptosis, and expression of proteins that regulate cell cycle progression were assessed by flow cytometry, annexin V-PE and 7-AAD staining, and Western blotting, respectively. Results: HER3-DXd inhibited the anchorage-independent growth of HER3+/ER+ cells by &amp;gt;50% at 5 nM and their colony formation at 5-25 nM (P&amp;lt;0.05). To maximize HER3-DXd’s antitumor efficacy, we performed high-throughput siRNA screening and identified ATR, CD247, RAB7A, UPK3A, ROCK2, SLC29A1, and WNT7A as potential synergistic targets. Among these targets, inhibiting ATR with siRNA or BAY1895344 showed the most synergistic effect with HER3-DXd in HER3+/ER+ BC cells. In contrast, no synergistic effect was observed with the combination of BAY1895344 plus patritumab or control ADC (IgG-DXd), suggesting its dependence on HER3-DXd-mediated delivery of DXd. The combination of HER3-DXd plus BAY1895344 reprogrammed cell cycle progression from G2/M arrest to G1 arrest by inhibiting both ATR/Chk1/cyclin A2/CDK2 and ATR/Chk1/cyclin E/CDK2 signaling. The combination also reduced expression of H2AX, an ATR substrate that contributes to DNA repair, but increased that of γH2AX, indicating the induction of DNA damage. HER3-DXd and BAY1895344 synergistically inhibited growth of HER3+/ER+ BC cells by inducing apoptosis. Conclusion: The combination of HER3-DXd plus ATR inhibitors has therapeutic potential for overcoming tamoxifen resistance in HER3+/ER+ BC. We are currently validating the synergy in endocrine-resistant ER+ BC xenograft models. Citation Format: Xuemei Xie, Jangsoon Lee, Jon A. Fuson, Huey Liu, Young Jin Gi, Pang-Dian Fan, Kumiko Koyama, Debu Tripathy, Naoto T. Ueno. Targeting ATR enhances the antitumor efficacy of patritumab deruxtecan (HER3-DXd) in tamoxifen-resistant ER+ breast cancer cells by reprogramming cell cycle progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB088.

Abstract CT572: Phase 1b dose-escalation and dose-expansion study evaluating trastuzumab deruxtecan (T-DXd) in combination with durvalumab and cisplatin, carboplatin, or pemetrexed in advanced or metastatic, HER2-overexpressing, nonsquamous non-small cell lung cancer (NSCLC): DESTINY-Lung03

Abstract Background: HER2 overexpression is an established molecular target in breast and gastric cancer therapy, but no HER2-targeted therapies are currently available for NSCLC. In studies of NSCLC and lung cancer overall, the prevalence of HER2 overexpression has been reported to be anywhere from &amp;lt;5% to 35% (Cox et al. Int J Cancer. 2001;92:480-483; Hirsch et al. Lancet. 2017;389:299-311). T-DXd is an antibody-drug conjugate composed of a humanized anti-HER2 monoclonal antibody, a tetrapeptide-based cleavable linker, and a topoisomerase I inhibitor payload. Interim results from the DESTINY-Lung01 trial showed encouraging efficacy in extensively pretreated patients (pts) with unresectable/metastatic, HER2-overexpressing, nonsquamous NSCLC, with a confirmed objective response rate (ORR) of 24.5% and median overall survival (OS) of 11.3 months (Nakagawa et al. WCLC 2020. Abstract OA04.05). Preclinically, the combination of T-DXd and an anti-programmed cell death 1 antibody was more effective than either therapy alone. The DESTINY-Lung03 trial is evaluating the safety and tolerability of T-DXd in combination with durvalumab and chemotherapy in pts with unresectable/metastatic, HER2-overexpressing, nonsquamous NSCLC. Methods: DESTINY-Lung03 (NCT04686305) is a phase 1b, open-label, multicenter, dose-escalation (part 1) and dose-expansion (part 2) study in pts with HER2-overexpressing (≥25% immunohistochemistry 2+/3+ tumor cells), unresectable, locally advanced or metastatic, nonsquamous NSCLC. In part 1, pts with progression after 1 or 2 lines of systemic therapy in the recurrent/metastatic setting are enrolled to receive T-DXd and durvalumab in combination with cisplatin (arm 1A), carboplatin (arm 1B), or pemetrexed (arm 1C), or T-DXd monotherapy (arm 1D). In part 2, pts who have not received prior curative treatment in the locally advanced setting or systemic treatment in the metastatic setting are enrolled to receive T-DXd and durvalumab in combination with cisplatin (arm 2A), carboplatin (arm 2B), or pemetrexed (arm 2C), or T-DXd plus durvalumab (arm 2D); pts in part 2 must not have activating EGFR mutations, an EML4-ALK fusion, or other targetable alterations. The primary endpoint is the frequency of adverse events (AEs) and serious AEs (SAEs) with T-DXd plus durvalumab plus chemotherapy combinations to assess safety and tolerability and determine the recommended phase 2 dose. Secondary endpoints include confirmed ORR, duration of response, disease control rate, progression-free survival, OS, frequency of AEs and SAEs with T-DXd monotherapy (part 1) and T-DXd plus durvalumab (part 2), pharmacokinetics, and immunogenicity. Citation Format: David Planchard, Julie R. Brahmer, James C. Yang, Yuh-Min Chen, Kang-Yun Lee, Thatthan Suksombooncharoen, Natasha Viglianti, Mark Gustavson, Alejandra Ragone, Amaya Gasco Hernandez. Phase 1b dose-escalation and dose-expansion study evaluating trastuzumab deruxtecan (T-DXd) in combination with durvalumab and cisplatin, carboplatin, or pemetrexed in advanced or metastatic, HER2-overexpressing, nonsquamous non-small cell lung cancer (NSCLC): DESTINY-Lung03 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT572.

Abstract 5287: Aggregation of HER2 receptors on the tumor cell surface induced by a novel anti-HER2 bispecific antibody led to enhanced tumor cell growth inhibition

Abstract Trastuzumab, an anti-HER2 monoclonal antibody developed by Roche, is now a standard of care for treating HER2 overexpressing cancers. However, patients treated with trastuzumab generally develop resistance to the drug within a year after initiation of the treatment. Pertuzumab, another anti-HER2 monoclonal antibody developed by Roche, targets the domain 2 of the HER2 excellular domain (ECD) instead of the domain 4 to which trastuzumab binds and demonstrates enhanced antitumor activities when combined with trastuzumab. Here, we developed a tetravalent bispecific antibody (BsAb) which targets the domain 2 and 4 of the HER2 ECD simultaneously. Using time-lapse microscopy, we showed that the BsAb induced aggregation of HER2 on NCI-N87 tumor cells, possibly indicating that the receptors could be crosslinked by the BsAb intermolecularly. Furthermore, the tetravalent BsAb induced aggregation faster than did the bivalent BsAb. Importantly, the tetravalent BsAbs exhibited superior tumor cell growth inhibition to that of the two parental monoclonal antibodies alone or combined. In line with the aggregation rates, the tetravalent BsAb was also more potent in inhibiting tumor cell growth than that of the bivalent BsAb. Thus we provide a piece of evidence that the BsAbs targeting two distinct domains of HER2 simultaneously might induce the receptor aggregation which might associate with the enhanced inhibition of tumor cell growth. Citation Format: Lan Deng, Jie Zhao, Xiaoqing Meng, Aotian Xiao, Zhenping Zhu, Haomin Huang. Aggregation of HER2 receptors on the tumor cell surface induced by a novel anti-HER2 bispecific antibody led to enhanced tumor cell growth inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5287.

Abstract 5376: Pleclinical investigation of T-DXd internalization and payload release in HER2 mutant non-small cell lung cancer cells

Abstract Introduction: T-DXd (trastuzumab deruxtecan) is an antibody-drug conjugate (ADC) targeting human epidermal growth factor receptor 2 (HER2) composed of a humanized anti-HER2 monoclonal antibody conjugated to a DNA topoisomerase I inhibitor, DXd through a linker. Internalization of HER2-T-DXd followed by DXd release is a crucial process for T-DXd to exert its antitumor activity. T-DXd induced robust and durable responses in HER2 mutant metastatic non-small cell lung cancer (NSCLC) patients in DESTINY-Lung01 study, including those with low HER2 expression levels (Li BT, et al. NEJM 2021). One hypothesis of this T-DXd sensitivity of HER2 mutant NSCLC cells involves the potential of enhanced receptor and ADC internalization in these cells (Li BT, et al. Cancer Discov 2020). To examine this possibility further, we investigated the effect of HER2 mutations on T-DXd internalization and DXd release in transgenic cells expressing various HER2 mutants, as well as on T-DXd sensitivity in vivo. Methods: We established transgenic NCI-H322 cells expressing wild-type (WT) HER2 or various HER2 mutants and evaluated T-DXd internalization in these cells. Antitumor activity of T-DXd was examined in xenograft mouse models of transgenic NCI-H322 cells and compared between WT and each mutant. DXd concentration in tumor was also determined. In addition, in vitro internalization activity and antitumor activity of T-DXd were evaluated using NCI-H1781, the NSCLC cell line harboring an endogenous HER2 mutation (G776delinsVC). Results: Transgenic NSCLC cells expressing various HER2 mutants showed higher T-DXd internalization than WT HER2 expressing cells. There also was a negative correlation between the surface HER2 expression level and T-DXd internalization among various mutants and WT expressing cells. In xenograft models with transgenic cells, some HER2 mutant models showed higher response to T-DXd than WT HER2 model, with greater than 2-fold increase in G660D, YVMA, G776delinsVC, V777_G778insGSP, and V842I. Tumor DXd concentration was positively correlated with the antitumor activity. In an endogenous HER2 mutant NSCLC cell line (NCI-H1781), T-DXd also showed high internalization activity and potent antitumor activity. Conclusion: In vitro study with transgenic NSCLC cells suggested that T-DXd internalization in HER2 mutant tumors was more efficient than in WT HER2 tumor. Some of those HER2 mutant cell lines showed increased T-DXd sensitivity and higher payload release in the tumor in vivo, suggesting the enhanced internalization played a role in the mechanism of T-DXd sensitivity in these HER2 mutant transgenic NSCLC cells. Some mutants may be more or less clinically relevant. However, the relationship between the in vitro internalization efficiency and in vivo antitumor activity in each mutant was not consistent among different mutants, thus these results need to be interpreted cautiously. Citation Format: Atsushi Yamasaki, Misa Ikuta, Manabu Abe, Tsuneo Deguchi, Yasuki Kamai, Kenji Nakamaru, Toshinori Agatsuma. Pleclinical investigation of T-DXd internalization and payload release in HER2 mutant non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5376.

The analgesic compound palmitoylethanolamide reduces inflammation in human cardiomyocytes and vascular endothelial cells exposed to doxorubicin and anti-HER2 monoclonal antibody through PPAR-α and NLRP3-related pathways.

e24054 Background: Palmitoylethanolamide (PEA) is an endogenous fatty acid mediator that is synthetized from membrane phospholipids by N-acyl phosphatidylethanolamine phospholipase D. It is a new analgesic drug with anti-inflammatory effects through the induction of PPAR-related pathways. We aimed to assess whether palmitoylethanolamide co-incubated during doxorubicin and trastuzumab, reduces anticancer drugs-related cardiotoxicity in cellular models. Methods: Human vascular endothelial cells and cardiomyocytes were exposed to subclinical concentration of doxorubicin (at 100 and 200 nM) combined to trastuzumab (at 100 and 200 nM) alone or in combination with a formulation composed by palmitoylethanolamide 500 nM for 48h. After the incubation period, we performed the following tests: determination of cell viability, through analysis of mitochondrial dehydrogenase activity, study of lipid peroxidation (quantifying cellular Malondialdehyde and 4-hydroxynonenal), intracellular Ca2+ homeostasis. Moreover, pro-inflammatory studied were also performed (activation of NLRP3 inflammasome; expression of peroxisome proliferator-activated receptor-α; mTORC1 Fox01/3a; transcriptional activation of p65/NF-κB and secretion of cytokines involved in cardiotoxicity (Interleukins 1β, 8, 6). Results: Palmitoylethanolamide co-incubated with doxorubicin exerts vasculoprotective and cardioprotective effects, enhancing cell viability of 56.3-78.7 % compared to untreated cells (p &lt; 0,001 for all). Notably, PEA reduced significantly the cardiotoxicity through peroxisome proliferator-activated receptor-α –related pathways and NLRP3 inflammasome but without the involvement of calcium homeostasis. Several cytokines and chemokines were also reduced confirming its anti-inflammatory effect. Conclusions: The present study demonstrates that palmitoylethanolamide protects against vasculotoxicity and cardiotoxicity of doxorubicin and trastuzumab by promoting an anti-inflammatory phenotype, representing a new therapeutic approach to resolve doxorubicin-induced vasculo-cardio toxicity and inflammation.

Results from the phase 1/2 study of patritumab deruxtecan, a HER3-directed antibody-drug conjugate (ADC), in patients with HER3-expressing metastatic breast cancer (MBC).

1002 Background: Patritumab deruxtecan (HER3-DXd) is a novel, investigational ADC composed of a human anti-HER3 monoclonal antibody covalently bound to a topoisomerase I inhibitor payload via a stable tetrapeptide-based cleavable linker. Here we report updated safety and efficacy data from this ongoing study (U31402-A-J101; NCT02980341; JapicCTI-163401) of HER3-DXd in pts with previously treated MBC. Methods: U31402-A-J101 is a phase 1/2, multicenter, open-label, first-in-human study of HER3-DXd in pts with HER3-expressing MBC (N = 182). The study enrolled pts in dose-escalation (3.2-8.0 mg/kg IV Q3W) and dose-finding portions across molecular subtypes (n = 66; including HER2+ MBC, n = 14) followed by dose expansion in the following subtypes: HER3 high (4.8 mg/kg [n = 33] or 6.4 mg/kg [n = 31]), HER3 low (6.4 mg/kg [n = 21]) HR+/HER2− MBC or HER3-high TNBC (6.4 mg/kg [n = 31]). HER3-high and -low were defined as ≥75% and 25% ‒ &lt; 75% membrane positivity. The primary objective was to assess safety and efficacy; secondary objectives included determining the relationship between efficacy and HER3 expression. Results: At data cutoff (16 Aug 2021), median study duration was 31.9 mo (range, 15-56). Median age was 57 y (range, 30-83); 132 (72.5%) and 50 (27.5%) pts had an ECOG PS of 0 or 1. Pts had a median of 5 (range, 1-13) prior lines of therapy for locally advanced/metastatic disease. Median treatment duration with HER3-DXd was 5.9 mo (range, 0.7-30.6). In a pooled evaluation of dose escalation/finding and expansion, efficacy is shown in pts with HR+/HER2− MBC, TNBC, and HER2+ MBC in the Table. Overall, 130 pts (71.4%) had grade ≥3 TEAEs; the most common (≥15%) were decreased neutrophil count (39.6%), decreased platelet count (30.8%), anemia (18.7%), and decreased white blood cell count (18.1%). 12 pts (6.6%) experienced treatment-related interstitial lung disease according to central adjudication, including 1 grade 5 event. Conclusions: A pooled analysis in this heavily pretreated population showed promising efficacy in pts with HR+/HER2− and HER2+ MBC as well as TNBC. The safety profile with longer follow-up is consistent with previous reports and showed adequate safety and tolerability. Studies are ongoing in MBC tumor types, with a focus on biomarkers associated with efficacy. Clinical trial information: NCT02980341. [Table: see text]

The effect of breast radiotherapy parameters (XRT), chemotherapy regimens (CT) and dual anti-HER2 monoclonal antibodies (MoAb) on the development of cardiac dysfunction (CD) among patients with HER2-positive breast cancer from Saudi Arabia.

e12508 Background: Treatment with anti-HER2 therapy can cause CD. The effect of breast XRT, heart dose and V25 of the heart and CT regimen on developing CD is controversial. The impact of these variables in developing CD among patients with HER2-positive breast cancer (BC), receiving CT plus anti-HER2 MoAb was evaluated in routine clinical practice. Methods: Patients with HER2-positive BC between 2015-2019 were included, retrospectively. CD is defined as a drop in (EF%) of at least 10 % points from baseline and/or below 50% at any point of the time during follow-up. The association of CD and various variables was examined with a significance level of &lt; 0.05. Results: Data from 224 female patients were analyzed with a mean age at diagnosis of 51 +12, BMI 29.6 ± 6.4, BSA 1.74 ± 0.19, 53% with left BC, 63% had non-metastatic BC, 52% with comorbidities, 11% had prior IHD medication use in 48%; metformin in 15% and B-blockers in 5%, 9.4% smoker, 70% had a mastectomy, 18% lumpectomy and 12% no surgery, 58% received XRT. The cumulative incidence of CD was 33% during follow-up noting that the first and subsequent drops of EF at any point of time during the follow-up was counted as a new event even for the same patient. The univariate analysis of factors influencing the risk of CD is shown in Table. However, the initial significance of CT regimen and whether the patient received breast XRT or otherwise, did not translate into significance on multivariate analysis with a P-value = 0.198 &amp; 0.326, respectively. Conclusions: XRT mean heart dose and V25 of the heart, anthracycline-based CT did not influence the risk of CD among this cohort. Likewise, the dual anti-HER2 therapy with trastuzumab and pertuzumab and the number of trastuzumab cycles did not influence CD. It is reassuring to demonstrate the safety of these approaches in real-life clinical practice.[Table: see text]

SO-7 Co-occurring HER2 and PD-L1 expression in patients with HER2-positive trastuzumab-refractory gastric cancer (GC)/gastroesophageal junction adenocarcinoma (GEJA): Biomarker analysis from the trastuzumab deruxtecan (T-DXd) DESTINY-Gastric03 trial

In up to 20% of patients (pts), GC/GEJA is HER2 positive. T-DXd is an antibody-drug conjugate composed of a humanized anti-HER2 monoclonal antibody, tetrapeptide-based cleavable linker, and topoisomerase I inhibitor payload. In the DESTINY-Gastric01 trial, T-DXd improved survival vs standard chemotherapy in HER2-positive GC/GEJA in the ≥3L setting. In previous reports, up to 88% of HER2-positive GC/GEJA tumors were PD-L1 positive (combined positive score [CPS] ≥1; Janjigian Nature 2021). Combined expression of HER2 and PD-1/PD-L1 in GC has been shown to have prognostic relevance (Lian Dig Liver Dis 2022), and preclinical data indicate improved efficacy of T-DXd in combination with an anti–PD-1 antibody.

Dose-finding and -expansion studies of trastuzumab deruxtecan in combination with other anti-cancer agents in patients (pts) with advanced/metastatic HER2+ (DESTINY-Breast07 [DB-07]) and HER2-low (DESTINY-Breast08 [DB-08]) breast cancer (BC).

3025 Background: Trastuzumab deruxtecan (T-DXd), an antibody-drug conjugate composed of a humanized anti-HER2 monoclonal antibody and a topoisomerase I inhibitor payload, is approved for pts with unresectable or metastatic HER2+ BC with ≥2 prior anti-HER2–based therapies. T-DXd showed improved progression-free survival vs trastuzumab emtansine (T-DM1) as an earlier-line treatment (tx) for pts with HER2+ metastatic BC in the phase 3 DESTINY-Breast03 trial (Cortes J, et al. Ann Oncol. 2021;32:S1283-S1346. Abstract LBA1). Preliminary antitumor activity of T-DXd was shown in heavily pretreated pts with HER2-low advanced/metastatic BC in the phase 1 DS8201-A-J101 trial (Modi S, et al. J Clin Oncol. 2020;38:1887-1896). We report preliminary results from the dose-finding phase of 2 trials investigating T-DXd combination tx in HER2+ or HER2-low metastatic BC. Methods: DB-07 (phase 1b/2; NCT04538742) and DB-08 (phase 1b; NCT04556773) are 2-part, modular, open-label, multicenter trials of T-DXd combined with other anticancer tx in pts with advanced/metastatic BC that is HER2+ (DB-07) or HER2 low (DB-08). Part 1 of each study is an ongoing dose-finding phase; pts must have ≥1 prior tx for metastatic BC. Part 2 of each study is a dose-expansion phase; pts must have no (DB-07) or ≤1 (DB-08) prior tx for metastatic BC. We report preliminary results from the T-DXd + pertuzumab module of DB-07 part 1 (data cutoff: Oct 15, 2021) and T-DXd + anastrozole and T-DXd + fulvestrant modules of DB-08 part 1 (data cutoff: Sep 27, 2021); pts in the DB-08 modules must be hormone receptor positive. The part 1 primary objective was to assess safety and tolerability and determine the recommended phase 2 dose (RP2D) according to the modified toxicity probability interval-2 algorithm. Pts were followed up beyond the 21-day dose-limiting toxicity (DLT) period (28 days for T-DXd + fulvestrant) for safety events. Results: In DB-07, 7 pts were enrolled and received T-DXd 5.4 mg/kg + pertuzumab 420 mg (loading dose: 840 mg) every 3 wk (q3w; not evaluable for DLTs, n = 1). In DB-08, 6 pts were enrolled and received T-DXd 5.4 mg/kg q3w + anastrozole 1 mg daily (not evaluable for DLTs, n = 1); another 6 pts were enrolled and received T-DXd 5.4 mg/kg q3w + fulvestrant 500 mg every 4 wk (loading dose: 500 mg cycle 1 days 1 and 15). For all 3 modules, no DLTs were reported in any DLT-evaluable pts; the dose levels used in part 1 were approved to be the RP2Ds for use in the dose-expansion part of each corresponding module. No deaths on study or cases of interstitial lung disease/pneumonitis were reported to date. Conclusions: The RP2Ds for the T-DXd combinations were the standard doses for BC of each individual drug. These studies are ongoing, with additional T-DXd combinations being evaluated and further follow-up underway. Clinical trial information: NCT04538742; NCT04556773.