anti-HER2 Antibody-drug Conjugate Research Articles

Combined therapy of dabrafenib and an anti-HER2 antibody–drug conjugate for advanced BRAF-mutant melanoma

BackgroundMelanoma is the most lethal skin cancer characterized by its high metastatic potential. In the past decade, targeted and immunotherapy have brought revolutionary survival benefits to patients with advanced and metastatic melanoma, but these treatment responses are also heterogeneous and/or do not achieve durable responses. Therefore, novel therapeutic strategies for improving outcomes remain an unmet clinical need. The aim of this study was to evaluate the therapeutic potential and underlying molecular mechanisms of RC48, a novel HER2-target antibody drug conjugate, either alone or in combination with dabrafenib, a V600-mutant BRAF inhibitor, for the treatment of advanced BRAF-mutant cutaneous melanoma.MethodsWe evaluated the therapeutic efficacy of RC48, alone or in combination with dabrafenib, in BRAF-mutant cutaneous melanoma cell lines and cell-derived xenograft (CDX) models. We also conducted signaling pathways analysis and global mRNA sequencing to explore mechanisms underlying the synergistic effect of the combination therapy.ResultsOur results revealed the expression of membrane-localized HER2 in melanoma cells. RC48 effectively targeted and inhibited the growth of HER2-positive human melanoma cell lines and corresponding CDX models. When used RC48 and dabrafenib synergically induced tumor regression together in human BRAF-mutant melanoma cell lines and CDX models. Mechanically, our results demonstrated that the combination therapy induced apoptosis and cell cycle arrest while suppressing cell motility in vitro. Furthermore, global RNA sequencing analysis demonstrated that the combination treatment led to the downregulation of several key signaling pathways, including the PI3K-AKT pathway, MAPK pathway, AMPK pathway, and FOXO pathway.ConclusionThese findings establish a preclinical foundation for the combined use of an anti-HER2 drug conjugate and a BRAF inhibitor in the treatment of BRAF-mutant cutaneous melanoma.

Abstract LB058: N-glycan deglycosylation ameliorates anti-HER2 antibody drug conjugate-induced interstitial pneumonia in preclinical models

Abstract Background: Current clinical application of trastuzumab deruxtecan (T-DXd), an anti-HER2 antibody-drug conjugate (ADC) has been severely limited by interstitial lung disease (ILD). Recent studies suggest a correlation between ILD and the uptake of T-DXd by alveolar macrophages (AMs), which are major innate immune cells in lung tissues. This underlying mechanism of AMs phagocytosing T-DXd was predicted to be the binding interaction between the crystallizable fragment (Fc) of trastuzumab and the Fcγ receptors on the cell surface of AMs, exerting effector functions. Since monoclonal antibodies typically undergo N-glycosylation in the Fc fragment, here we investigate the impacts of antibody N-glycan deglycosylation on T-DXd-induced ILD in preclinical models. Methods: N-glycan removal was performed on trastuzumab and T-DXd using PNGase F. Using immunofluorescence imaging combined with flow cytometry, we conducted a comparative analysis of the binding affinity and internalization efficiency of wild type and fully N-glycan deglycosylated HER2 antibodies. Next, we determined the in vitro cytotoxicity of WT and N-glycan deglycosylated T-DXd against both macrophages and HER2-positive cancer cells using CCK-8 assay. Furthermore, the deposition of N-glycan deglycosylated T-DXd in healthy lungs and their effects in ILD incidence and severity was determined in immunocompetent BALB/c mice carrying HER2-positive tumors. Results: First, we obtained complete N-glycan deglycosylated HER2 antibodies confirmed by SDS-PAGE and mass spectrometry. Next, we showed that N-glycan removal by PNGase F did not affect the Fab fragments of HER2 antibodies binding to the HER2 antigens on HER2-expressing cancer cells (OE19, SK-OV-3 and CT26-hHER2). Importantly, we did observe an approximately 30-50% reduction in the N-glycan deglycosylated HER2 antibodies binding with a panel of three macrophages (MH-S, RAW264.7, and PMA-induced THP-1) in comparison with WT antibodies, indicating a significant impact of N-glycan removal on innate immune cell-mediated effector functions. Moreover, in vitro cytotoxicity studies further support these observations that N-glycan deglycosylated T-DXd has significantly lower cytotoxicities against MH-S (p=0.046) and RAW264.7(p=0.020) in comparison with WT T-DXd. Correlating with our in vitro findings, in vivo biodistribution data showed a reduced lung deposition of HER2 antibodies and T-DXd after N-glycan removal. Immunofluorescence staining confirms the co-localization of HER2 antibodies with CD68-positive macrophages, indicating that AMs are the major contributor to the lung deposition of HER2 antibodies and T-DXd and N-glycan removal could effectively avoid ADC being phagocytosed by AMs. We also demonstrated chronic administration of N-glycan deglycosylated T-DXd could reduce ADC-associated ILD incidence and ameliorate ILD severity. Conclusions: Collectively, our study suggests that antibody N-glycan deglycosylation has important roles in regulating interactions between ADC and innate immune cells. This finding provides valuable clues for further optimizing ADC design and treatment strategies. Citation Format: Yu-Xuan Yang, Le-Tao Ma, Peng Guo. N-glycan deglycosylation ameliorates anti-HER2 antibody drug conjugate-induced interstitial pneumonia in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB058.

Abstract 2111: Impact of HER2 Expression on trastuzumab deruxtecan(T-DXd) efficacy

Abstract Background: T-DXd is an FDA-approved anti-HER2 Antibody Drug Conjugate (ADC), which has shown clinical efficacy in breast cancers with either overexpression or low-level expression of HER2. The benefit of T-DXd across the spectrum of HER2 expression has called into question the relevance of HER2 expression level as relevant parameter for T-DXd sensitivity. We sought to experimentally address the relevance of HER2 expression level upon the degree of T-DXd response. Methods: A series of isogenic models was generated that expressed different levels of surface HER2 using a doxycycline-inducible promoter. These were analyzed for impact of differential expression level upon T-DXd internalization and drug response. Human breast cancer samples were analyzed for HER2 expression levels and association with clinical response to T-DXd under an IRB approved protocol. Results: Induction of different levels of HER2 at specified doxycycline concentrations was verified in multiple models using immunoblotting and FACS. These different levels appeared to correspond to clinical breast cancer immunohistochemistry (IHC) scoring levels to 0, 1+, 2+, and 3+. The models were analyzed for T-DXd response and showed increasing levels of HER2 led to increase lysosomal uptake of T-DXd (p<0.0001). Moreover, elevation of HER2 levels led to lower concentrations of T-DXd needed to suppress tumor growth. Analyses of the effects of increasing levels of HER2 upon xenograft response to T-DXd are ongoing. Finally, retrospective analyses of a cohort of 50 breast cancer patients treated with T-DXd revealed that higher levels of HER2 corresponded to longer time on treatment (time to progression). Conclusion: We observe a consistent relationship between elevated expression of HER2 and breast cancer response to T-DXd. The findings highlight the relevance of HER2 expression level as a biomarker of T-DXd response and support the potential value of therapeutic strategies that can induce HER2 surface expression levels to enhance response. Citation Format: Wanyi Chen, Joshua Drago, Avantika Gupta, Bo Liu, Sarat Chandarlapaty. Impact of HER2 Expression on trastuzumab deruxtecan(T-DXd) efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2111.

Abstract 6788: Comparative analysis of anti-HER2 antibody-drug conjugates in tumor spheroid models

Abstract Robust techniques are needed to screen the anti-tumor activity of antibody-drug conjugates (ADCs) in vitro. Traditional monolayer models provide a cost-effective and scalable option but lack many features of the tumor microenvironment, such as complex cell-cell and cell-extracellular matrix interactions. Tumor spheroids afford a more translational model for quantification of ADCs. Here, spheroids are used to compare the mechanism of action (MoA) of anti-HER2 ADCs. Single spheroids were formed from mono- or co-cultures of HER2 over-expressing cancer cells and HER2 negative control cells. Incucyte® Nuclight Green Lentivirus was used to label HER2 positive BT474 cells, whilst HER2 negative MDA-MB-231 cells were left unlabeled. Antibodies were added after 72 hours of spheroid formation in 96-well ultra-low attachment plates. Brightfield and fluorescence images were captured every 3 hours using the Incucyte® Live-Cell Analysis System and quantified, for metrics such as spheroid size and fluorescence intensity, to give an indication of ADC cytotoxicity. Co-culture spheroids were dissociated at assay endpoint and the remaining proportion of green and unlabeled cells was measured using the iQue® Advanced Flow Cytometry Platform. The area of the BT474 mono-culture spheroids decreased in a concentration-dependent manner in the presence of both ADCs tested (trastuzumab emtansine and trastuzumab deruxtecan), indicating increased cytotoxicity. In contrast, only the highest concentration of trastuzumab, which is the monoclonal antibody backbone on which they are based, resulted in a reduction in spheroid size. This displays the increased anti-tumor activity due to the addition of the ADC payloads. In the co-culture model, only the trastuzumab deruxtecan induced a reduction in spheroid area, with the size of the spheroids in the presence of trastuzumab emtansine and trastuzumab remaining comparable to control levels. Analysis of the spheroid composition using flow cytometry showed that for the BT474 cells, again, percentage cell death compared to the IgG control was greater for the two ADCs, at 96.4% and 97.2%, respectively. This death was greater than with trastuzumab, which induced 46.4% cell death. Unlike in the mono-culture assays, a high level of death of the HER2 low expressing cells was also seen in the co-culture in the presence of trastuzumab deruxtecan (94.1% death compared to control). This displays the unique bystander activity of trastuzumab deruxtecan, which has led to its approval as a treatment for both HER2 high and HER2 low breast cancers. These data display how the Incucyte® Live-Cell Analysis System combined with iQue® Advanced Flow Cytometry can be leveraged to provide comprehensive assessment of the in vitro function of ADCs and how it can enable differences in their MoAs to be distinguished in 3D spheroid models. Citation Format: Kirsty McBain, Kalpana Barnes, Nicola Bevan. Comparative analysis of anti-HER2 antibody-drug conjugates in tumor spheroid models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6788.

HER2-low Breast Cancer: Now and in the Future.

Breast cancer is a heterogeneous disease, and its subtypes are characterized by hormone receptor and human epidermal growth factor receptor 2 (HER2) expression status. "HER2-low" tumors, which exhibit a low level of HER2 expression (immunohistochemistry 1+ or 2+ without gene amplification), were conventionally considered not amenable to anti-HER2 targeting agents based on the results of a phase III trial of trastuzumab. However, this perspective is being challenged by the emergence of novel anti-HER2 antibody-drug conjugates, such as trastuzumab-deruxtecan. These innovative therapies have demonstrated remarkable efficacy against HER2-low breast cancer, shedding new light on a previously overlooked category of breast cancer. Such promising results highlight the need for in-depth investigations of the biology and prognostic implications of HER2-low tumors. In this review, we comprehensively summarize the current evidence surrounding this topic and highlight areas that warrant further exploration and research in the future.

Systemic Therapies for HER2-Positive Advanced Breast Cancer.

Despite recent advances, HER2-positive advanced breast cancer (ABC) remains a largely incurable disease, with resistance to conventional anti-HER2 drugs ultimately unavoidable for all but a small minority of patients who achieve an enduring remission and possibly cure. Over the past two decades, significant advances in our understanding of the underlying molecular mechanisms of HER2-driven oncogenesis have translated into pharmaceutical advances, with the developing of increasingly sophisticated therapies directed against HER2. These include novel, more potent selective HER2 tyrosine kinase inhibitors (TKIs); new anti-HER2 antibody-drug conjugates; and dual epitope targeting antibodies, with more advanced pharmacological properties and higher affinity. With the introduction of adjuvant T-DM1 for incomplete responders to neoadjuvant therapy, fewer patients are relapsing, but for those who do relapse, disease that may be resistant to standard first- and second-line therapies requires new approaches. Furthermore, the risk of CNS relapse has not been abrogated by current (neo)adjuvant strategies; therefore, current research efforts are being directed towards this challenging site of metastatic disease. In this article, we review the currently available clinical data informing the effective management of HER2-positive breast cancer beyond standard first-line therapy with pertuzumab, trastuzumab, and taxanes, and the management of relapse in patients who have already been exposed to both these agents and T-DM1 for early breast cancer (EBC). We additionally discuss novel anti-HER2 targeted agents and combinations in clinical trials, which may be integrated into standard treatment paradigms in the future.

Prevalence and Therapeutic Targeting of High-Level ERBB2 Amplification in NSCLC

IntroductionERBB2 amplification in lung cancer remains poorly characterized. HER2 (encoded by ERBB2) is a transmembrane tyrosine kinase capable of ligand-independent dimerization and signaling when overexpressed, and a common cause of HER2 overexpression is ERBB2 amplification. Here, we evaluated the clinicopathologic and genomic characteristics of ERBB2-amplified NSCLC and explored a HER2 antibody–drug conjugate (ADC) therapeutic strategy. MethodsOur institutional next-generation DNA sequencing data (OncoPanel) from 5769 NSCLC samples (5075 patients) were queried for cases having high-level ERBB2 amplification (≥6 copies). Clinical and demographic characteristics were extracted from the electronic medical records. Efficacy of the pan-ERBB inhibitor afatinib or HER2 ADCs (trastuzumab deruxtecan and trastuzumab emtansine) was evaluated in NSCLC preclinical models and patients with ERBB2 amplification. ResultsHigh-level ERBB2 amplification was identified in 0.9% of lung adenocarcinomas and reliably predicted overexpression of HER2. ERBB2 amplification events are detected in two distinct clinicopathologic and genomic subsets of NSCLC: as the sole mitogenic driver in tumors arising in patients with a smoking history or as a concomitant alteration with other mitogenic drivers in patients with a light or never smoking history. We further reveal that trastuzumab deruxtecan is effective therapy in in vitro and in vivo preclinical models of NSCLC harboring ERBB2 amplification and report two cases of clinical activity of an anti-HER2 ADC in patients who acquired ERBB2 amplification after previous targeted therapy. ConclusionsHigh-level ERBB2 amplification reliably predicts HER2 overexpression in patients with NSCLC, and HER2 ADC is effective therapy in this population.

Generation of a Novel SORT1×HER2 Bispecific Antibody-Drug Conjugate Targeting HER2-Low-Expression Tumor.

Human epidermal growth factor receptor 2 (HER2) is considered an ideal antibody-drug conjugate (ADC) target because the gene is overexpressed in many tumors compared to normal tissues. Multiple anti-HER2 ADCs conjugated with different toxic payloads bring benefits to patients with high HER2 expression. However, HER2-targeted ADC technology needs further optimization to improve its effect for the treatment of patients with low HER2 expression. We hypothesized that bispecific antibody-drug conjugate (bsADC) targeting HER2 and Sortilin-1 (SORT1) would overcome this limitation. SORT1 is a suitable target for pairing with HER2 to generate a bispecific antibody (BsAb) since the gene is co-expressed with HER2 in tumors and possesses rapid internalization. We developed a BsAb (bsSORT1×HER2) that exhibited strong binding and internalization activity on HER2-low-expression tumor cells and facilitated higher HER2 degradation. The bsSORT1×HER2 was further conjugated with DXd to generate a bsADC (bsSORT1×HER2-DXd) that showed strong cytotoxicity on HER2-low-expression tumor cells and antitumor efficacy in an MDA-MB-231 xenograft mice model. These results demonstrated that employment of a SORT1×HER2-targeted bsADC may be promising to improve the antitumor efficacy of HER2-targeted ADC for the treatment of tumors with low HER2 expression.

Clinical characteristics and treatment outcomes of HER2 mutation and HER2 fusion in 22 patients with advanced breast cancer.

The clinical characteristics and efficacy of human epidermal growth factor receptor-2 (HER-2)-directed agents against HER2 mutations and HER2 fusions in breast cancer are obscure due to their low frequency. We conducted a retrospective study in patients with advanced breast cancer harboring HER2 mutations and/or HER2 fusions between January 1, 2017 and January 1, 2021. Among a total of 22 patients, 17 HER2 mutations were detected, including L755S, S310F, R100=, V777L, R897W, T862A, 440-17C > G, H878Y, V842I, 73 + 9G > C, T278fs, E1069K, L755P, 226-11C > T, 574 + 12C>T, L114V and P128L. The majority of patients had ductal carcinoma, which mostly coexisted with HER2 amplification/overexpression. The median progression-free survival (PFS) of the 22 patients was 6.9 months (95% CI: 4.7, 9.1) in the first-line setting. The median PFS of patients who received first-line trastuzumab-based regimens was significantly longer than that of patients who received a first-line tyrosine kinase inhibitor (TKI) (10.8 months [95% CI: 2.9, 18.7] vs. 1.9 months [95% CI: 0.8, 3.0], p < 0.005). A total of 14 patients were treated with anti-HER2 antibody-drug conjugate (ADC), among whom the median treatment line of first-time of administration of anti-HER2 ADC was 4.5 (range, 1-10). Anti-HER2 ADC reached an objective response rate (ORR) of 42.9%, a disease control rate (DCR) of 85.7% and a median PFS of 7.3 months (95% CI: 4.4-10.1) from the first-time of administration. Our data demonstrated the clinical benefit of anti-HER2 treatment in Chinese breast cancer patients harboring HER2 mutation and/or HER2 fusion. The value of immunotherapy and treatment selection among individual HER2 variants needs further study.

An Innovative Regimen Basing on HFRT/SBRT and RC48-ADC Coactivation for Salvage Therapy in Patients with HER2-Expressing Advanced Solid Tumors

It is now widely accepted that radiotherapy, especially hypofractionated radiation therapy (HFRT) or stereotactic radiotherapy (SBRT), can modulate tumor phenotypes, enhance antigen presentation and provoke a systemic immune response which gives a strong rationale for the combination of RT and immunotherapy (iRT). The PRaG therapy is an innovative iRT, when combined with HFRT/SBRT, PD-1/L1 inhibitor and GM-CSF to activate the immune response and modulate the tumor microenvironment to exert the desired in abscopal effect. Previous studies have demonstrated encouraging efficacy of the PRaG regimen in the treatment of advanced refractory tumors. RC48-ADC is a promising anti-HER2 antibody-drug conjugate with inducing immunogenic cell death and widespread release of cancer cell antigens, synergize with immunotherapy by promoting effector T-cell activation. The aim of this study is to explore efficacy and safety of RC48-ADC combined with radiotherapy, PD-1/L1 inhibitor sequential GM-CSF and IL-2(PRaG3.0 regimen) for treatment of HER2-expressing advanced solid tumors. Participants with advanced, confirmed HER2-expressing (IHC3+, 2+ or 1+) solid tumors that had progressed after standard treatment, or intolerance were enrolled. In a PRaG3.0 regimen cycle, those received RC48-ADC (2.0 mg/kg d1, every 3 weeks), then HFRT (2-3 doses of 5-8 Gy) was delivered for one metastatic lesion every other day, followed by GM-CSF (200 μg d3-7), sequential IL-2(2million IU d8-12), and PD-1/L1 inhibitor was dosing within one week after completion of HFRT. After RC48-ADC combined with PD-1/L1 inhibitor sequential GM-CSF and IL-2 for at least 6 cycles, then maintenance with PD-1/L1 inhibitor was administered until disease progression or unacceptable toxicity. The primary endpoint was objective response rate (ORR). This trial is registered with ClinicalTrials.gov, number NCT05115500. With the cutoff date of 31 December 2022, a total of 30 patients (n = 6 for gynecological cancer, n = 5 for pancreatic cancer, n = 19 for other cancers) were enrolled, in which 21 patients completed at least 1 tumor assessment. The objective response rate (ORR) was 42.9%, and the disease control rate was 71.4% by RECIST1.1. The ORR was 66.7% in gynecological cancer, 25.3% in pancreatic cancer, and 36.4% in other cancers. Median progression-free survival (PFS) for all patients was 7.0 months (95% CI: 3.4, 10.7). The most common treatment-related adverse events (TRAEs) included fatigue, fever, alopecia and anorexia. Grade ≥3 TRAEs occurred in two patients (6.7%). These preliminary results show that of PRaG3.0 regimen has a manageable safety profile and encouraging antitumor activity in heavily pretreated patients with HER2- expressing cancers. Ultimately the regimen achieved the accurate integration of RT, immunotherapy and targeted therapy.

Evaluating Low HER2 Status in Invasive Breast Carcinoma via HER2 Immunohistochemistry, with HER2 FISH Correlation: A Cohort of 112 Patients.

Recent trials demonstrated clinically significant benefits in HER2-nonamplified breast cancer with HER2-low expression using novel anti-HER2 antibody-drug conjugates. Thus, HER2-low breast cancer was proposed as a separate diagnostic entity. Herein, we reclassify HER2-negative cancers according to the new HER2-low category using a modified system and further investigate HER2-very-low expression. 114 HER2 immunohistochemistry (IHC)-negative invasive breast tumors were identified from the pathology database of Mayo Clinic, Jacksonville, FL, between January 2019 and August 2022. Two blinded breast pathologists (BP) independently rescored HER2 IHC slides at 200x and 400x magnification. Discordant cases between the two BPs were rescored together. The most recent 2018 ASCO/CAP HER2 scoring criteria were used. HER2 (0) was subdivided into HER2 (absent) and HER2 (very low). HER2 FISH testing was performed in all cases. The cohort comprised of 38 (33.3%) HER2 (0) and 76 (66.7%) HER2 (1+) tumors. The first round of rescoring at 200x and 400x magnification resulted in 17 (14.9%) HER2 (absent), 31 (27.2%) HER2 (very low), and 64 (56.2%) HER2 (1+) and 2 (1.8%) HER2 (2+) tumors by BP1 and 20 (17.5%) HER2 (absent), 33 (28.9%) HER2 (very low), and 61 (53.5%) HER2 (1+) tumors by BP2. The combined final rescoring by BP1 and BP2 was as follows: 15 (13.2%) HER2 (absent), 35 (30.7%) HER2 (very low), 63 (55.3%) HER2 (1+), and 1 (0.9%) HER2 (2+) cases. A comparison of the first round of rescoring between two BPs showed substantial agreement with Cohen's kappa value of 0.67. Both comparisons of first rescoring by BP1 and by BP2 to combined final rescoring showed almost perfect agreement with Cohen's kappa value of 0.83.Follow-up FISH studies showed one amplified tumor. Our data support the need for finer granularity, classification, and understanding of HER2-low breast cancers. We also show that reproducibility between trained BP can be obtained, albeit with scoring at high power and low threshold for showing challenging interpretations.

Development, methodological evaluation and application of a cell-based TRF assay for analysis of ADCC activity

Interaction of an antibody with its FcγR plays an important role in effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC). Nowadays altered ADCC activity of an antibody can be achieved by utilizing an effective glyco-engineering strategy, which often involves changes of sugar moieties in Fc part of the antibody, thereby affecting its receptor binding with effector cells. We aimed to construct a cell-based time-resolved fluorescence (TRF) assay for the evaluation of ADCC activity triggered by the antibody drug Trastuzumab (anti-HER2) and T-DM1. The assay was initiated by incubating 2,2′:6′,2 "-Terpyridine-6,6"-dicarboxylic acid (TDA)-labeled target SK-BR3 cells with the testing antibodies and engineered NK-92 effector cells. After incubation, the target cells were lysed to detect TDA released into the supernatant. Together with added Eu, the TDA in the supernatant formed a stable chelate of EuTDA with high-intensity fluorescence. The ADCC activity was then determined by measuring the fluorescence of EuTDA. Consequently, the method demonstrated good accuracy, precision, linearity, and specificity over methodological assessment and compared well with the Luciferase release assay in terms of the agreement of the achieved results. Using the developed assay, we evaluated the ADCC activity of two glyco-engineered anti-HER-2 antibody-drug conjugates (ADCs) and the results showed that antibody Fc glycosylation modifications influenced antibody ADCC activity to varying degrees. In conclusion, the present assay is able to accurately assess the ADCC activity induced by Trastuzumab (anti-HER2) and T-DM1, and a similar methodology can be applied to other therapeutic antibodies during drug development to help screen for antibodies with desirable ADCC activity.

Clinicopathological characteristics of HER2-low breast cancer: a retrospective study

Human Epidermal Growth Factor Receptor-2 (HER2)-negative breast cancers (BCs) contain HER2-low and HER2-zero ones. HER2-low breast cancer has been receiving wide-spread concerns as the marvelous effect of novel anti-HER2 antibody-drug conjugates, however, the characteristic remains unknown. Our aim was to explore the differences of clinicopathological indicators and survival outcomes between HER2-low and HER2-0 breast cancers. We retrospectively analyzed 501 invasive breast cancer patients with complete data on HER2 status from 2017 to 2021 in our single center, of whom 415 HER2 negative patients were included for subsequent analysis. Each cohort was further divided into hormone receptor (HR) positive and HR negative subgroup. Clinicopathological factors and survival outcomes were collected and compared between HER2-low BCs and HER2-0 BCs. HER2-low BCs was obviously higher in HR positive BCs, with 277 (90.5%) HER2-low HR positive patients, 29 (9.5%) HER2-low HR negative patients, 68 (62.4%) HER2-0 HR positive patients and 41 (37.6%) HER2-0 HR negative patients (P < 0.001). Significant differences between HER2-low BCs and Her2-0 BCs were observed in lymph node ratio (LNR) (mean rank, 215 vs. 188 P = 0.014), estrogen receptor (ER)expression (90.5% vs. 62.4% P < 0.001), progesterone receptor (PR) expression (84.3% vs. 56.9% P < 0.001), Ki-67 expression (46.4% vs. 61.5% P < 0.001), androgen receptor (AR) expression (68% vs. 50.5% P < 0.001), adjuvant chemotherapy (69% vs. 79.8% P = 0.03). HER2-low BCs had lower histological grade than HER2-0 BCs, with grade I–II (68.7% vs. 43.1%) and grade III (22.2% vs. 43.1%) P < 0.01. No statistical differences were detected between the two groups for DFS and DDFS. Our results demonstrated that HR and AR status was closely related to HER2-low breast cancers. Further exploration about survival prognosis of HER2-low breast cancer is badly needed.

HER2-low breast cancer: evolution of HER2 expression from primary tumor to distant metastases

BackgroundBreast cancer (BC) with low human epidermal growth factor receptor 2 (HER2) expression is attracting much attention due to the breakthrough progress of novel anti-HER2 antibody-drug conjugates. HER2 expression is examined in patients with HER2-low BC and their distant metastases in this study, so as to further clarify the dynamic characteristics of HER2 low status in the process of disease progression.MethodsPatients diagnosed with HER2 low breast cancer (defined as IHC1+ or IHC2+/ISH-) between 2012 and 2021 were included in this study. We evaluated HER2 expression of primary sites and metastatic sites, compared the impact of different clinicopathological parameters on HER2 status of metastases and compared the overall survival and disease-free survival of patients with different HER2 status in metastases.ResultsNinety-eight patients were included. All HER2 IHC scores were confirmed and the consistent rate with the original pathological report was 81.1%. 27.6% of the patients showed different HER2 status in metastases. The HER2 discordance rate differed among different metastatic sites (p = 0.040). The higher the T stage of the primary BC, the higher the rate of HER2 discordance was observed (p = 0.042). For the specimen type of metastasis, HER2 discordant rate was higher in surgical specimen than biopsy (p = 0.050). No difference of HER2 discordance rate was found between HER2-1+ and HER2-2+ patients. But comparing HER2 IHC score, HER2-2+ patients were less likely to have consistent metastatic HER2 levels than HER2-1+ patients (p = 0.006). No difference in survival outcomes was observed between patients with different HER2 status in metastases.ConclusionsThere is a possibility of HER2 expression alteration in the metastases of HER2-low breast cancer. And the rate of altered HER2 low expression was different among different metastatic sites, different T stages of primary BC and specimen type of metastasis. No prognostic significance was observed.

Analytical comparability to evaluate impact of manufacturing changes of ARX788, an Anti-HER2 ADC in late-stage clinical development.

ARX788 is an anti-HER2 antibody drug conjugate (ADC) developed using Ambrx proprietary Engineered Precision Biologics technology. The manufacturing process of ARX788 has been optimized during the course of early to late-phase clinical development. A comprehensive evaluation of side-by-side comparability between pre- and post-change process for ARX788 drug substance and drug product from a quality perspective was conducted based on ICH Q5E guidelines consisting of batch release assays, physicochemical and biophysical characterization, biological characterization, and forced degradation studies. All results have substantiated a high degree of similarity between the pre- and post-change ARX788 drug substance batches and drug product lots, demonstrating that the process manufacturing changes did not impact product quality.

A review of treatment options in HER2-low breast cancer and proposed treatment sequencing algorithm.

The expansion of the spectrum of human epidermal growth factor receptor 2 (HER2)-status to HER2-low, defined as HER2 expression of 1+ by immunohistochemistry (IHC) or 2+ by IHC without gene amplification, has made a major impact in the field of oncology. The HER2-low expression has emerged as a targetable biomarker, and anti-HER2 antibody-drug conjugate trastuzumab deruxtecan has shown significant survival benefit in pretreated metastatic HER2-low breast cancer (BC). With these recent data, the treatment algorithm for hormone receptor-positive and triple-negative BC needs to be reconsidered, as approximately half of these BCs are HER2-low. Although there are different therapeutic agents for hormone receptor-positive and hormone receptor-negative HER2-low BCs, there is no consensus regarding the sequencing of these agents. In this article, the treatment options for HER2-low BC are enumerated and a treatment sequencing algorithm based on the current clinical evidence proposed.

Molecular correlation of response to pyrotinib in advanced NSCLC with HER2 mutation: biomarker analysis from two phase II trials

BackgroundNon-small cell lung cancer (NSCLC) with HER2 mutation has entered into the era of targeted therapy. However, both anti-HER2 antibody–drug conjugates (ADCs) and tyrosine kinase inhibitors (TKIs) showed moderate objective response rate (ORR) and median progression-free survival (PFS). The aim of this study was to investigate the molecular features of responders to pyrotinib in advanced NSCLC with HER2 mutation.MethodsPatients from our two previous phase II trials were pooled analyzed. Their circulating tumor DNA (ctDNA) were detected by next-generation sequencing (NGS) panels, and the correlation with the efficacy of pyrotinib was investigated.ResultsThis pooled analysis included 75 patients, and 50 of them with baseline plasma samples were finally enrolled with a median age of 57 years old. The overall ORR and median PFS were 28% and 7.0 months respectively. Biomarker analysis showed that 5 patients were ctDNA nonshedding. Patients with TP53 wild type were significantly associated with higher disease control rate (97.1%vs. 68.8%, p = 0.010), PFS (median 8.4 vs. 2.8 months, p = 0.001) and overall survival (OS, median 26.7 vs. 10.4 months, p < 0.001) than those with mutations. ctDNA of nonshedding and clearance exhibited significantly longer PFS (median: 10.2 vs. 9.8 vs. 5.6 months, p = 0.036) and a trend of longer OS (median: 35.3 vs. 18.1 vs. 14.6 months, p = 0.357) than those not.ConclusionPatients with TP53 wild type, ctDNA nonshedding, or clearance showed superior efficacy of pyrotinib in patients with HER2-mutated advanced NSCLC, which might be helpful to guide the utility of pyrotinib in clinical setting.Trial registration: The patients were from two registered clinical trials (ClinicalTrials.gov: NCT02535507, NCT02834936).

Assessment of HER2 status in extramammary Paget disease and its implication for disitamab vedotin, a novel humanized anti-HER2 antibody-drug conjugate (ADC) therapy.

e21544 Background: Evaluation of HER2 status in extramammary Paget disease (EMPD) has been reported in a few studies with a small number of samples, and the HER2 overexpression rate ranges from 15% to 60%. The prognosis of advanced EMPD is poor and the treatment options is limited. Disitamab Vedotin (DV) is a novel humanized anti-HER2 antibody-drug conjugate (ADC), has been approved for metastatic urothelial carcinoma and gastric cancer in China. To determine the HER2 status and the efficacy of DV in advanced EMPD patients (pts) harboring HER2 expression, we conducted this multicenter retrospective study. Methods: Paraffin-embedded blocks, clinic-pathological data, and survival data of EMPD pts were retrospectively collected from 3 centers in China. HER2 IHC scoring and gene amplification evaluation were carried out according to the (ASCO/CAP) 2018 guidelines. The results of FISH and IHC were analyzed by two pathologists independently in a double-blinded manner. Data analysis was performed using SPSS v.26, Prism 8 and R version 3.6.5. Results: Finally, 129 cases with detailed data were enrolled between 2006 and 2022. Among them, HER2 IHC was performed in 102 cases, and FISH in 46 cases. HER2 expression was detected in 93.1% samples, with scores of 1+, 2+, and 3+ in 47.1% (48/102), 37.3% (38/102), and 8.7% (9/102) respectively. HER2 gene amplification was detected in eight cases (8/46, 17.4%), including all the cases with IHC 3+ (5/5, 100%), two with IHC 2+ (2/18, 11.1%), and one with IHC 1+ (1/22, 4.5%). For the subgroup of HER2 2+/3+, a higher frequency of lymph node metastasis (66.7% vs 33.3%, P = 0.034) and reduced survival time were detected than in HER2 1+/0. Two elderly male pts with advanced EMPD (one HER1+ /FISH-, one HER 2+/FISH-), who failed with chemo of paclitaxel, received 4 doses of DV infusion (2mg/kg every 2 weeks), and achieved partial responses both with tolerable toxicity. Conclusions: According to the study, the expression rate of HER-2 was very high in EMPD, indicating that it may be used as a routine treatment target. DV showed efficacy in these patients and need further validation.

FS-1502 in HER2-positive advanced breast cancer: Results from an open-label, phase 1 study.

3044 Background: FS-1502 is an anti-HER2 antibody-drug conjugate that was well tolerated and showed good antitumor activity in patients (pts) with advanced HER2 -expressing solid tumors in the dose-escalation stage of the current Phase 1 study. The results were presented at SABCS 2022. The recommend Phase 2 dose (RP2D) was 2.3 mg/kg IV once every 3 weeks. Here, we report the efficacy and safety of HER2-positive breast cancer (BC) pts treated with FS-1502 at the RP2D. Methods: This open-label Phase 1 trial was conducted at 9 hospitals in China. Heavily pretreated HER2-positive (IHC 3+ or 2+/FISH+) BC pts who had failed prior anti-HER2 therapy were recruited in the dose-expansion stage. All HER2-positive BC pts treated at the RP2D were pooled for analysis. Efficacy endpoints included objective response rate (ORR) and disease control rate (DCR) per RECIST v1.1. Safety endpoints included treatment-emergent adverse events (TEAEs) and the laboratory data. Results: As of cutoff date, December 24, 2022, the median study follow-up duration was 5.2 months. A total of 70 female HER2-positive BC pts, with a median age of 51.8 (range: 31-76) years, were enrolled and received at least one dose of FS-1502. The median prior therapy line was 3 (range: 1-16), and 91.4% (64/70) of pts had previously received at least 2 therapy lines. The most common metastasis organs were lung 55.7% (39/70), lymph node 54.3% (38/70) and bone 50% (35/70). 81.4% (57/70) of pts had at least 3 metastatic lesions. All pts had previously received anti-HER2 therapy, among which trastuzumab ± pertuzumab (n = 67, 96%) and pyrotinib (n = 52, 74%) were the most common. Among 68 efficacy-evaluable pts, the best ORR of 52.9% (36/68) (95% CI, 40.45-65.17) was achieved with 2 complete responses and 34 partial responses. DCR was 88.2% (60/68), median progression-free survival was 15.5 months (95% CI, 4.57,-), and median duration of response was 12.9 months (95% CI, 5.72,-). FS-1502 was well tolerated. No related interstitial lung disease occurred in any grade. Eye toxicity was mostly Grade 1 or 2 of dry eye and keratitis. Grade ≥ 3 study-related TEAEs were reported in 27 (38.6%) pts, in which the most common events (≥ 5%) were hypokalemia (18.6%), platelet count decreased (7.1%), and neutrophil count decreased (5.7%). One (1.4%) patient experienced adverse events leading to drug discontinuation (hemoptysis). Adverse events leading to death were reported in 2 (2.9%) pts (bacterial pneumonia and hemoptysis, respectively). Bacterial pneumonia was not related to FS-1502, while hemoptysis was possibly related to the drug. Conclusions: FS-1502 demonstrated very promising antitumor activity and was well tolerated in heavily pretreated HER2-positive BC, with mild eye toxicity and no related interstitial lung disease reported. A Phase 3 clinical trial is under planning to further confirm efficacy and safety of FS-1502 in pts with advanced HER2-positive BC. Clinical trial information: NCT03944499 .

A multicenter, phase II trial of RC48-ADC combined with radiotherapy, PD-1/PD-L1 inhibitor, GM-CSF, and sequential IL-2 (PRaG3.0 regimen) for salvage therapy in patients with HER2-expressing advanced solid tumors.

e14614 Background: Cancer immunotherapy has emerged as one of the most promising approaches in many kinds of advanced tumors. The PRaG therapy is an innovative cancer immunotherapy, when combined with radiotherapy, PD-1/L1 inhibitor and GM-CSF to generate the desired in situ vaccine effect to activate the immune response and modulate the tumor microenvironment. Previous studies have showed encouraging efficacy of the PRaG regimen in the treatment of advanced refractory tumors. RC48-ADC, an anti-HER2 antibody-drug conjugate with MMAE as cytotoxic payload has been recently demonstrated that ADCs are also able to induce immunogenic cell death and widespread release of cancer cell antigens, synergize with immunotherapy by promoting effector T-cell activation. The aim of this study is to explore efficacy and safety of PRaG3.0 regimen for therapy of HER2-expressing advanced solid tumors. Methods: Participants with advanced, confirmed HER2-expressing (IHC3+, 2+ or 1+) solid tumors that had progressed after standard of care, or intolerance were enrolled. In the cycle, those received RC48-ADC (2.0 mg/kg d1, every 3 weeks), then HFRT (2-3 doses of 5-8Gy) was delivered for one metastatic lesion every other day, followed by GM-CSF (200 μg d3-7), sequential IL-2 (2million IU d8-12), and PD-1/L1 inhibitor was dosing within one week after completion of HFRT. After RC48-ADC combined with PD-1/L1 inhibitor sequential cytokines for at least 6 cycles, then maintenance with PD-1/PD-L1 inhibitor was administered until disease progression or unacceptable toxicity. The primary endpoint was objective response rate (ORR). Results: As of Jan 2023, a total of 32 patients (n=6 for gynecological cancer, n=5 for pancreatic cancer, n=21 for other cancers) were enrolled, 26 of them completed at least 1 tumor assessment. The ORR was 38.5%, and the disease control rate was 69.2%. The ORR was 66.7% in gynecological cancer, 25.0% in pancreatic cancer, and 31.3% in other cancers. Notably, patients who were HER2 IHC1+ responded similarly to those who were IHC2+~3+, with ORR of 43.8% and 30.0%, respectively. Median progression-free survival was 7.2 months (95%CI: 4.9, 9.5). The most common treatment-related adverse events (TRAEs) included fatigue (28.1%), fever (28.1%), alopecia (28.1%) and anorexia (18.8%). Grade ≥3 TRAEs occurred in two patients (6.3%). In addition, activated plasmacytoid dendritic cell was significantly higher at baseline in responders (CR+PR) vs. nonresponders (SD+PD). Conclusions: The schedule of RC48-ADC was changed from once every 2 weeks to once every 3 weeks, and was still effective with significantly reduced side effects. These preliminary results suggest that PRaG3.0 regimen has a manageable safety profile and enhancing potential sensitivity in pretreated patients with HER2-expressing cancers. Clinical trial information: NCT05115500 .