HER2-targeting Antibody-drug Conjugate Research Articles

A DXd/TLR7-agonist dual-conjugate anti-HER2 ADC exerts robust anti-tumor activity through tumor cell killing and immune activation.

The emergence of trastuzumab deruxtecan (T-DXd), a new-generation antibody-drug conjugate (ADC), has profoundly altered the therapeutic paradigm for HER2-positive solid tumors, demonstrating remarkable clinical benefits. However, the combined outcomes of T-DXd with immunotherapy agents remain ambiguous. In this study, we introduce Tras-DXd-MTL1, an innovative HER2 targeting ADC that integrates the topoisomerase inhibitor DXd and a toll-like receptor 7 (TLR7) agonist MTT-5, linked to trastuzumab via a GGFG tetrapeptide linker. Mechanistically, Tras-DXd-MTL1 retains the DNA-damaging and cell-killing properties of topoisomerase inhibitors while simultaneously enhancing the immune response within the tumor microenvironment (TME). This is achieved by promoting immune cell infiltration and activating dendritic cells and CD8+T cells via MTT-5. In vivo evaluation of Tras-DXd-MTL1's anti-tumor potency revealed a notably superior performance compared to the T-DXd (Tras-DXd) or Tras-MTL1 in immunocompetent mice with trastuzumab-resistant EMT6-HER2 tumor and immunodeficient mice with JIMT-1 tumor. This improved efficacy is primarily attributed to its dual functions of immune stimulation and cytotoxicity. Our findings highlight the potential of incorporating immunostimulatory agents into ADC design to potentiate antitumor effects and establish durable immune memory, thereby reducing tumor recurrence risks. Therefore, our study offers a novel strategy for the design of immune-activating ADCs and provides a potential approach for targeting solid tumors with different levels of HER2 expression.

The Evolving Paradigm of Antibody-Drug Conjugates Targeting the ErbB/HER Family of Receptor Tyrosine Kinases.

Current therapies targeting the human epidermal growth factor receptor (HER) family, including monoclonal antibodies (mAbs) and tyrosine kinase inhibitors (TKIs), are limited by drug resistance and systemic toxicities. Antibody-drug conjugates (ADCs) are one of the most rapidly expanding classes of anti-cancer therapeutics with 13 presently approved by the FDA. Importantly, ADCs represent a promising therapeutic option with the potential to overcome traditional HER-targeted therapy resistance by delivering highly potent cytotoxins specifically to HER-overexpressing cancer cells and exerting both mAb- and payload-mediated antitumor efficacy. The clinical utility of HER-targeted ADCs is exemplified by the immense success of HER2-targeted ADCs including trastuzumab emtansine and trastuzumab deruxtecan. Still, strategies to improve upon existing HER2-targeted ADCs as well as the development of ADCs against other HER family members, particularly EGFR and HER3, are of great interest. To date, no HER4-targeting ADCs have been reported. In this review, we extensively detail clinical-stage EGFR-, HER2-, and HER3-targeting monospecific ADCs as well as novel clinical and pre-clinical bispecific ADCs (bsADCs) directed against this receptor family. We close by discussing nascent trends in the development of HER-targeting ADCs, including novel ADC payloads and HER ligand-targeted ADCs.

Preclinical studies of BB-1701, a HER2-targeting eribulin-containing ADC with potent bystander effect and ICD activity.

Several HER2-targeting antibody-drug conjugates (ADC) have gained market approval for the treatment of HER2-expressing metastasis. Promising responses have been reported with the new generation of ADCs in patients who do not respond well to other HER2-targeting therapeutics. However, these ADCs still face challenges of resistance and/or severe adverse effects associated with their particular payload toxins. Eribulin, a therapeutic agent for the treatment of metastatic breast cancer and liposarcoma, is a new choice of ADC payload with a distinct mechanism of action and safety profile. We've generated a novel HER2-tageting eribulin-containing ADC, BB-1701. The potency of BB-1701 was tested in vitro and in vivo against cancer cells where HER2-expressing levels vary in a large range. Bystander killing effect and toxin-induced immunogenic cell death (ICD) of BB-1701 were also tested. In comparison with HER2-targeting ADCs with DM1 and Dxd payload, eribulin-containing ADC demonstrated higher in vitro cytotoxicity in HER2-low cancer cell lines. BB-1701 also effectively suppressed tumors in models resistant to DM1 or Dxd containing ADCs. Mode of action studies showed that BB-1701 had a significant bystander effect on HER2-null cells adjacent to HER2-high cells. In addition, BB-1701 treatment induced ICD. Repeated doses of BB-1701 in nonhuman primates showed favorable pharmacokinetics and safety profiles at the intended clinical dosage, route of administration, and schedule. The preclinical data support the test of BB-1701 in patients with various HER2-expressing cancers, including those resistant to other HER2-targeting ADCs. A phase I clinical trial of BB-1701 (NCT04257110) in patients is currently underway.

Trastuzumab deruxtecan (DS-8201a), a HER2-targeting antibody-drug conjugate, demonstrates in vitro and in vivo antitumor activity against primary and metastatic ovarian tumors overexpressing HER2.

High-grade serous ovarian cancer (HGSOC) and ovarian clear cell carcinoma (CC), are biologically aggressive tumors endowed with the ability to rapidly metastasize to the abdominal cavity and distant organs. About 10% of HGSOC and 30% of CC demonstrate HER2 IHC 3 + receptor over-expression. We evaluated the efficacy of trastuzumab deruxtecan (T-DXd; DS-8201a), a novel HER2-targeting antibody-drug conjugate (ADC) to an ADC isotype control (CTL ADC) against multiple HGSOC and CC tumor models. Eleven ovarian cancer cell lines including a matched primary and metastatic cell line established from the same patient, were evaluated for HER2 expression by immunohistochemistry and flow cytometry, and gene amplification by fluorescence in situ hybridization assays. In vitro experiments demonstrated T-DXd to be significantly more effective against HER2 3 + HGSOC and CC cell lines when compared to CTL ADC (p < 0.0001). T-DXd induced efficient bystander killing of HER2 non-expressing tumor cells when admixed with HER2 3 + cells. In vivo activity of T-DXd was studied in HER2 IHC 3 + HGSOC and CC mouse xenograft models. We found T-DXd to be significantly more effective than CTL ADC against HER2 3 + HGSOC (KR(CH)31) and CC (OVA10) xenografts with a significant difference in tumor growth starting at day 8 (p = 0.0003 for KR(CH)31, p < 0.0001 for OVA10). T-DXd also conferred a survival advantage in both xenograft models. T-DXd may represent an effective ADC against primary and metastatic HER2-overexpressing HGSOC and CC.

HER2 expression-independent antitumor effect of trastuzumab deruxtecan (T-DXd) on pediatric solid tumors.

e15016 Background: Treatment of pediatric solid tumors shows limited improvement even with intensification of conventional chemotherapy, suggesting novel approaches such as targeted therapies are needed. Trastuzumab Deruxtecan (T-DXd), a HER2-targeting antibody–drug conjugate, exhibits significant anti-cancer activity in breast cancers with a broad range of HER2 expression. T-DXd might be a promising agent for refractory or relapsed pediatric solid tumors, but its efficacy in pediatric tumors needs to be studied. Methods: Anti-cancer efficacy of T-DXd and its payload DXd were assessed in vitro by utilizing our in-house cancer cell line models. Cytotoxicity assays were performed for 60 pediatric cancer cell lines, including Neuroblastoma (NB) = 25, Ewing sarcoma / Ewing sarcoma family of tumors (EWS / EWSFT) = 17, Rhabdomyosarcoma (RMS) = 10, Others (Osteosarcoma, Brain tumor, Wilms tumor, Hepatoblastoma, and Malignant rhabdoid tumor (MRT) = 8, and 2 breast cancer cell lines expressing HER2 high or low (IHC score 2+ or 0) as positive or low control, respectively. We analyzed HER2 expression on the cell surface by flow cytometry and evaluated relative mean fluorescence intensity (MFI) values. Results: In the flow cytometric assessment, pediatric cancer cell lines showed smaller median MFI of 1.40 (0.67 - 2.71) compared with breast cancer control with HER2 high expression (33.77). The median MFI of each cell lines were 0.94 in NB, 1.76 in EWS / EWSFT, 1.42 in RMS, 1.52 in other pediatric cancers. Although low or no expression of HER2, certain inhibitory activities to the cell growth were observed for T-DXd against a part of NB, EWS, RMS and MRT cell lines, with the minimum IC50 values of 14.5 nM, 6.5 nM, 10.2 nM, 8.1 nM, respectively. In NB, EWS and RMS, a wide range of sensitivity to T-DXd were seen among different cell lines in the same disease. Most of cell lines showed high sensitivity to DXd (Median IC50 value: 0.90 nM [0.08 - 6.46 nM]), but, among them, cell lines with lower sensitivity to DXd tend to show less sensitivity to T-DXd. There was no clear correlation between sensitivity to T-DXd and HER2 expression among pediatric cancer cell lines, indicating that other mechanism such as intrinsic sensitivity to payload might affect antitumor activity of T-DXd. Conclusions: T-DXd exhibited antitumor efficacy against pediatric solid tumors in vitro irrespective of HER2 expression. Our results suggest that T-DXd might be an alternative therapy for pediatric solid tumor cases for whom conventional chemotherapies and other targeting therapies are ineffective. This study supports further investigation for T-DXd in this population.

Identification of HER2 ultra-low based on an artificial intelligence (AI)-powered HER2 subcellular quantification from HER2 immunohistochemistry images.

1115 Background: HER2-targeted antibody-drug conjugates (ADCs) can effectively target tumor cells even in HER2-low breast cancers (1+ and 2+/no amp). We developed an AI-based whole-slide image (WSI) analyzer for immunohistochemistry (IHC)-stained slides, exploring a cutoff differentiating between true HER2 negative versus HER2 ultra-low. Methods: An AI-based IHC WSI analyzer was developed using 6,188 WSIs stained by 19 types of IHC including HER2 and from various cancers, annotated by 153 pathologists. The model predicts cells as tumor cells or non-tumor cells and generates HER2 expression continuous score (HER2ecs, 0-100%), quantifying IHC staining intensity of membrane, nucleus, cytoplasm, and membrane+cytoplasm separately for each cell. A total of 401 HER2 IHC WSIs were acquired from Samsung Medical Center (n=275), Kyung Hee University Hospital (n=78), and Commercial Biobank (n=48), scored as 0 (n=347) and 1+ (n=54) by pathologists. Results: In total, 67,169,114 tumor cells and 119,113,886 non-tumor cells from 401 WSIs were successfully evaluated. Comparing HER2 score 0 versus 1+ by pathologists, mean HER2ecs of nucleus (1.7% vs 4.1%), cytoplasm (6.5% vs 12.9%), and membrane (3.6% vs 8.2%) were comparable. In HER2 score 0 WSIs, membrane HER2ecs of tumor cell was 3.6 ± 7.8% (mean ± standard deviation), while that of non-tumor cell was 0.9 ± 2.9% (p &lt;0.001 vs. tumor cell), suggesting, even in HER2 score 0 cases, tumor cell exhibits HER2 expression beyond non-specific HER2 staining intensity. Based on our internal validation dataset, membrane-specific HER2ecs of 6.0% was the optimal threshold discriminating HER2 no-staining cells versus HER2 incomplete and faint/barely perceptible cells (AI-HER2-weak) per ASCO/CAP guideline. Among HER2 score 0 by pathologists, when applying this cutoff, the proportion of WSIs showing AI-HER2-weak or higher membrane-specific HER2ecs in over 10% of all tumor cells was 23.6% (82/347). Interestingly, that of WSIs among HER2 score 1+ by pathologists was 51.9% (28/54), which is comparable to 52.3%, objective response rate of trastuzumab deruxtecan in HER2-low patients in DESTINY-Breast04. Conclusions: AI-powered subcellular-level analysis enables identification of 23.6% HER2 ultra-low WSIs among HER2 score 0 by pathologist, which is defined by AI-HER2-weak or higher membrane-specific HER2 expression ≥ 10%. Clinical validation of the AI-powered HER2 ultra-low definition is warranted.

Efficacy and safety of disitamab vedotin (RC48) in HER2-expressing breast cancer: A multicenter retrospective study.

e13011 Background: RC48 is an Antibody-Drug Conjugate (ADC) that integrates a humanized monoclonal antibody targeting human epidermal growth factor receptor 2 (HER2) linked to the microtubule inhibitor monomethyl auristatin E (MMAE). This study reports real-world data using RC48 monotherapy in HER2-expressing advanced breast cancer (ABC). Methods: The study reviewed patients with HER2-expressing ABC who received RC48 monotherapy at three hospitals in China between September 2021 and June 2022. The electronic medical record was used to collect the clinical characteristics of the patient and to evaluate the antitumor activity and safety. The primary outcome was real-world progression-free survival (rwPFS). Results: A total of 15 patients were included, of whom 9(60%) were HER2-positive and 6(40%) had HER2 low expression (IHC 1+ or IHC 2+/ISH-).11 patients (73.3%) had visceral metastases, 40% of patients (6/15) had central nervous system (CNS) metastases at baseline. 6 patients (40%) had ≥3 metastatic lesions, and 13(86.7%) of patients received RC48 as third-line treatment or higher. Progression-free survival (PFS), overall survival (OS), and response can be assessed in all patients. The median follow-up was 10 months and the median rwPFS was 8.0 months (95% CI, 4.897-11.103), while the median OS was not reached. Objective response rate (ORR) and disease control rate (DCR) were 46.7% and 80% respectively. Partial response (PR) was achieved in 46.7% of the patients (7/15). patients with HER2-positive and HER2 low expression, the median rwPFS was 8.77 and 4.63 months, respectively (HR = 0.49,95% Cl, 0.13-1.80, P = 0.196), and in 6 patients with CNS metastases, the median rwPFS was 7.5 months (95% CI, 2.82-18.28). The most common treatment-related adverse events (TRAEs) were elevated transaminase (33.3%) and Leukopenia (33.3%), most of which were grade 1-2, No serious adverse events related to treatment were observed. Conclusions: The novel HER2-targeted ADC, RC48, demonstrated promising preliminary antitumor activity in patients with HER2-expressing ABC, patient with brain metastases could also benefit, with tolerable toxicities.

Clinical significance of HER2 expression between primary tumors and metastatic lymph nodes in patients with extramammary Paget's disease.

e21568 Background: HER2 has been reported to be a potential oncogene and therapeutic target in extramammary Paget's disease (EMPD). However, the expression of HER2 in EMPD, especially in matched metastatic lymph nodes (LNs), is limited. Here, we investigated the heterogeneity of HER2 expression between primary tumors and positive nodes and its clinical significance in advanced EMPD patients and detected the efficacy of HER2-targeted antibody drug conjugates (ADCs) in EMPD. Methods: A total of 170 patients with pathologically confirmed EMPD of the scrotum and/or penis treated at SYSUCC from 2003 to 2023 were included. HER2 IHC staining (anti-HER2/neu (4B5), Ventana) and scoring were performed according to the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) 2023 guidelines. Outcome relationships were explored using Cohen's kappa coefficient, multivariate Cox regression and log-rank analysis. Results: A total of 102 primary tumor and 32 metastatic specimens were ultimately subjected to HER2 IHC staining. HER2 high-expression (IHC: 2/3+) was detected in 71.6% (0: 9.8%, 1+: 18.6%, 2+: 40.2% and 3+: 31.4%) and 68.8% (0: 21.9%, 1+: 9.4%, 2+: 28.1% and 3+: 40.6%) of the primary tumors and metastatic LNs, respectively. HER2-high EMPD patients experienced poorer outcomes, with a 5-year OS of 56.5%, than did HER2-low patients (P = 0.032). A multivariate Cox regression model showed that a positive lymph node (HR 15.39, 95% CI 3.79~62.43; P &lt; 0.001) and HER2 high-expression in primary tumors (HR 3.68, 95% CI 1.49~9.09; P = 0.005) were independent poor prognostic factors. Notably, in 31 paired samples, 35.5% (n = 11) of patients had inconsistent HER2 expression between primary tumors and corresponding lymph node metastases. Six (19.4%) patients exhibited a shift from high expression of HER2 in primary tumors to low expression of HER2 in the lymph nodes, and 5 (16.1%) patients exhibited the reverse change. Advanced EMPD patients with HER2 high-expression were treated with Disitamab Vedotin (RC48), for which the ORR was 80% and the DCR was 100% (n = 5). No adverse events (AEs) above grade 3-4 were observed. Conclusions: HER2 was highly expressed in both primary and metastatic LNs in EMPD patients. However, there was some heterogeneity in HER2 expression between paired primary and corresponding metastatic LNs. The expression status of HER2 should be evaluated in both primary tumors and corresponding lymph node metastases. A positive lymph node and HER2 high-expression are poor prognostic factors in patients with EMPD. RC48 demonstrated significant efficacy in treating patients with HER2-high advanced EMPD and has promising potential that requires further validation.

A single-arm, multicenter, phase 2 clinical study of recombinant humanized anti-HER2 monoclonal antibody-MMAE conjugate (MRG002) in HER2-positive unresectable locally advanced or metastatic urothelial carcinoma

BackgroundMRG002 is a novel HER2-targeted antibody-drug conjugate being investigated in the MRG002–006 trial to evaluate the efficacy and safety in HER2-positive urothelial carcinoma patients. MethodsThis is an open-label, single-arm, multicenter phase II study. Eligibility criteria included: histologically confirmed HER2 IHC 2 + or 3 + UC, prior received ≥ 1 standard treatment. Patients in this study received MRG002 every 3 weeks until progressive disease or unacceptable toxicity. The primary endpoint was confirmed ORR per RECIST 1.1. ResultsAs of February 24, 2023, a total of 43 patients were enrolled. The median age was 60. 9 patients were dosed at 2.6 mg/kg and 34 patients were dosed at 2.2 mg/kg. At baseline, most patients (29/43) received ≥ 2 lines of treatment and 35 (81.4%) patients had prior ICI therapy. FISH test was performed in 41 patients and 9 (22.0%) were positive. By the cut-off date, 41 patients were evaluable and the ORR was 53% (95%CI：38.9%−67.5%), with 6.9% CR, and the DCR was 83.7% (95%CI：70.0%−91.9%). The median PFS and OS for the 43 patients were 7.0 months (95%CI：5.4-NE) and 14.9 months (95%CI：11.9-NE), respectively. The ORR was 77.8% in 9 patients with positive HER2 FISH results. Most common treatment-related AEs were anemia (51.2%), alopecia (44.2%) and neutropenia (39.5%); most were grade 1 or 2. ConclusionPreliminary results of MRG002 demonstrated a clinically meaningful response in pretreated HER-2 positive unresectable locally advanced or metastatic UC patients. MRG002 at 2.2 mg/kg was well tolerated with a manageable toxicity.

Abstract PS11-06: Analysis of HER2 expression changes from breast primary to brain metastases including HER2 Low and impact on overall survival

Abstract Background: Previous studies have shown that breast cancer receptor subtype switching between matched primary and metastatic brain metastases (BrM) is common. HER2 expression at low levels, termed “HER2 Low”, has emerged as a new therapeutic biomarker for highly active antibody drug conjugates with potential intracranial activity. Trastuzumab deruxtecan is FDA approved for the treatment of HER2 Low and HER2 positive breast cancer with emerging evidence of CNS responses and clinical benefit in patients with BrM. This study aimed to investigate the relationship between HER2 expression including HER2-Low between matched breast cancer primary tumors and brain metastases. We also sought to evaluate the impact of low HER2 expression on overall survival in breast cancer brain metastases. Methods: We identified pts with MBC seen at least once at a single, NCI-designated center and had a diagnosis of BrM between 2003 and 2023. Of 1252 MBC pts with a diagnosis of BrM, 265 pts underwent resection/biopsy for BrM with available ER/PR/HER2 status. Only pts with available matched primary and BrM tissue were analyzed. HER2 expression was defined as: HER2+ (3+ or 2+/ISH amplified), HER2-Low (1+, 2+/ISH neg) or HER2-0 [by ASCO-CAP guidelines]. Estrogen receptor (ER) status was defined as ER≥1%. Multivariate overall survival (OS) analyses by Cox proportional hazard models were determined from time of BrM resection to death or last follow-up between HER2+, HER2-Low and HER2-0 patients controlling for ER and age. Results: 197 matched breast cancer primary and BrMs with available ER, PR, and HER2 testing were evaluated. Median age at BrM resection was 51 yrs. ER was positive in 48% (N=95) of BrMs. Of 265 resected BrMs, HER2 expression was found in 72%: 49.8% HER2+ (N=132), 22.2% HER2-Low (N=59), and 27.9% HER2-0 (N=74). HER2 expression by 197 primary tumors was: 57% HER2+ (N=112), 24% HER2-Low (N=48), 19% HER2 0 (N=37). Amongst 112 HER2+ primary tumors, 97% (N=109) had concordant HER2+ BrMs, 3 (2.6%) switched to HER2-Low, with 100% retaining some HER2 expression. Of 37 HER2-0 primaries, 35% (13/37) gained HER2-Low expression and 5.4% (2/37) gained HER2+ expression in the BrM. Amongst 48 HER2-Low primary tumors, half (52%) changed expression to either HER2+ (21%) or HER2-0 (31.2%) in the brain. 66 tumors had extracranial tissue biopsied prior to BrM resection available for HER2 analysis. Of 15 HER2-0 extracranial tumors, 47% gained HER2-Low and 13% gained HER2+ at time of BrMs. After adjusting for age and ER status at the time of BrMs, patients with HER2+ BrM had a statistically significant lower risk of death at time of follow up than those with HER2-Low BrM (HR=0.41, P=0.0006). The risk of death between patients with HER2-0 BrM did not differ from HER2-Low BrM after adjustment for ER and age (HR=0.96, p =0.9). Median survival from BrM resection was 12.4m, 16.1m, 47.6m for HER2-Low, HER2-0 and HER2+ respectively (p = .0004). Conclusions: HER2 expression amongst primary, extracranial and brain metastases is dynamic with frequent change. Over 40% of HER2-0 primary or extracranial tumors gain HER2 expression in the brain. These changes have therapeutic implications given HER2 targeting antibody drug conjugates with improved OS and emerging evidence of CNS activity, highlighting the need for better diagnostics, including those that do not require invasive tissue sampling, to determine HER2 status in the brain. Patients with HER2-Low and HER2-0 BrMs have inferior survival and are a clinical unmet need. Citation Format: Alyssa Pereslete, Melissa Hughes, Alyssa Patterson, Janet Files, Kyleen Nguyen, Lauren Buckley, Ashka Patel, Abigail Moore, Eric Winer, Tianyu Li, Sara Tolaney, Nancy Lin, Sarah Sammons. Analysis of HER2 expression changes from breast primary to brain metastases including HER2 Low and impact on overall survival [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS11-06.

Abstract PO4-14-12: Predicting Response to HER2 Tyrosine Kinase Inhibitors and Antibody Drug Conjugates in HER2 Mutant Invasive Lobular Carcinoma Using CRISPR/Cas9 Knock-in Cell lines and Patient-derived Organoids

Abstract Background: Activating mutations in ERBB2 (HER2) are enriched &amp;gt;4-fold in invasive lobular carcinoma (ILC) with a rate of up to 19% in metastatic ILC. ILC is a histologic subtype of breast cancer characterized by loss of E-cadherin (CDH1), suggesting a potential interaction between loss of CDH1 and mutations in ERBB2. Recent trials have demonstrated promising single agent efficacy using the irreversible pan-HER tyrosine kinase inhibitor (TKI), neratinib, in patients with metastatic ERBB2 mutant ILC. However, further studies on combination therapies with other anticancer agents are needed to increase response rate and progression free survival for these patients. HER2-targeted antibody drug conjugates (ADC), particularly trastuzumab deruxtecan (T-DXd), have shown great promise in HER2-low metastatic breast cancer. Yet, their efficacy as a single agent or with HER2 TKIs in HER2-low and -mutant ILC is unknown and warrants investigation. Methods: Past studies analyzing ERBB2 mutations used overexpression of ERBB2 mutant cDNA, but this approach does not faithfully recapitulate the human disease. To model ERBB2 missense mutations as found in human breast cancers, we used CRISPR-based prime editing to generate a panel of isogenic ILC cell lines and patient-derived organoids (PDO) harboring ERBB2 wild-type (WT) or ERBB2 mutations (S310F or V777L). Both of these ERBB2 mutations have been previously characterized and are known to be activating mutations. We then used them to test neratinib and other TKIs with ADCs, including T-DXd and trastuzumab emtansine (T-DM1). Results: We successfully introduced single copy, heterozygous activating ERBB2 mutations (S310F or V777L) into two ERBB2-nonamplified metastatic ILC cell lines (MDA-MB-134 and SUM44PE) and one ERBB2-nonamplified metastatic ILC PDO (IPM-BO-053). Positive clones carrying the mutations were verified by Sanger sequencing and droplet digital PCR and subsequently pooled together. We further demonstrated that these mutations hyperactivated HER2 and downstream signaling pathways. ILC cell lines harboring these mutations showed enhanced sensitivity to HER2 TKIs but not ADCs. In contrary, ERBB2 mutations did not alter responses of IPM-BO-053 PDOs to HER2 TKIs but significantly increased responses to ADCs. Interestingly, we also observed accelerated HER2 protein degradation upon heregulin stimulation in ERBB2 mutant ILC PDOs, suggesting activating ERBB2 mutations may enhance ADC/HER2 complex internalization, degradation, and release of payloads. Lastly, we explored drug synergy between HER2 TKIs and ADCs in isogenic IPM-BO-053 PDOs and found that combination of T-DXd and neratinib or afatinib showed synergy (Combination Index &amp;lt; 1). Conclusions: Although the reason for the discrepancies in drug response between ILC cell lines and PDOs is not clear, we hypothesize that response in 3D PDOs might be more faithfully representing response seen in patients. We will generate additional ILC PDOs with knock-in ERBB2 mutations to validate our findings. Irreversible HER2 TKIs, such as neratinib and afatinib, showed synergy with T-DXd in ERBB2 mutant ILC PDOs. This holds important therapeutic implications in light of current treatment options for ILC. In future experiments, we will test if neratinib or afatinib increases endocytic uptake of T-DXd using a fluorescently labeled endocytosis tracker. Further, an in-depth molecular characterization of our isogenic cell lines and PDOs models is ongoing to gain mechanistic insights into how activating ERBB2 mutations increase HER2 internalization and degradation. Our in vitro studies provide a strong foundation for in vivo testing of neratinib or afatinib with T-DXd for ERBB2 mutant ILC. Citation Format: Jie Bin Liu, Danielle Tseng, Jagmohan Hooda, Daniel Brown, Adrian Lee, Steffi Oesterreich. Predicting Response to HER2 Tyrosine Kinase Inhibitors and Antibody Drug Conjugates in HER2 Mutant Invasive Lobular Carcinoma Using CRISPR/Cas9 Knock-in Cell lines and Patient-derived Organoids [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-14-12.

Abstract PO3-18-11: A011801 (CompassHER2 RD): Postneoadjuvant T-DM1 + tucatinib/placebo in patients with residual HER2-positive invasive breast cancer

Abstract Background: Patients with HER2+ early breast cancer (EBC) and invasive residual disease (RD) after neoadjuvant therapy (NAT) have a higher risk of relapse than patients who have a pathologic complete response (pCR). Escalation of therapy in patients with RD using post-neoadjuvant T-DM1 has become the new standard of care, leading to improved invasive disease-free survival (iDFS), but patients with estrogen receptor (ER)-negative or nodal RD have suboptimal outcomes, and central nervous system recurrences are a challenge. More effective treatment strategies are urgently needed. Alliance A011801 (CompassHER2 RD) is an escalation trial for patients with high-risk HER2+ RD after neoadjuvant systemic therapy, evaluating the addition of the HER2 selective tyrosine kinase inhibitor (TKI) tucatinib to post-neoadjuvant T-DM1. Methods: Eligibility and Intervention: Patients with high-risk HER2+ RD (i.e., ER-, node-positive, or both) after a predefined course of neoadjuvant HER2-directed treatment are randomized 1:1 to adjuvant T-DM1 + placebo, vs. T-DM1 and tucatinib with adjuvant RT +/- ET. Eligibility criteria include completion of ≥ 6 cycles of NAT, including ≥ 9 weeks of paclitaxel and trastuzumab +/- pertuzumab. All chemotherapy (CT) must be completed preoperatively unless participating in EA1181 (~15-30% will be participants in the CompassHER2 pCR de-escalation companion trial [NCT04266249]; these patients must receive postoperative CT to complete ≥ 6 cycles prior to enrollment on A011801). Patients who received prior HER2-targeted TKIs or antibody-drug conjugates are ineligible. Objectives: The primary objective is to determine if iDFS is improved with addition of tucatinib to T-DM1 in patients with HER2+ EBC with RD after neoadjuvant systemic therapy; secondary endpoints include overall survival, breast cancer free survival, distant recurrence-free survival, brain metastases-free survival and disease-free survival. Correlative objectives include the association of i) tumor infiltrating lymphocyte (TILs) levels in the primary tumor and RD with iDFS, ii) TILs with tucatinib benefit, iii) iDFS and circulating tumor cells (CTC) at serial timepoints and iv) the magnitude of benefit of tucatinib (iDFS) in patients with/without detectable pretreatment CTCs. Quality of life and pharmacokinetic endpoints are also being evaluated. Statistics: A011801 is a prospective, double-blind, randomized, phase III superiority trial; stratified by i) receipt of postoperative CT (Y/N), ii) hormone receptor-status (+/-), and iii) pathologic lymph node status (+/-). The study targets an absolute difference of 5% in iDFS (control vs. experimental arm 82% &amp; 87%, HR = 0.7), with a two-sided alpha of 0.05 and power of 80%. The sample size is 981; target accrual = 1031 patients; activation and estimated completion dates are 01/6/21 and ~ 01/2028. Accrual as of 7/10/2023: 438 patients. Support: U10CA180821, U10CA180882; Seagen Inc; https://acknowledgments.alliancefound.org. ClinicalTrials.gov Identifier: NCT04457596 Citation Format: Ciara O'Sullivan, Karla Ballman, Linda McCall, Tyler Zemla, Anna Weiss, Melissa Mitchell, Victoria Blinder, Nadine Tung, William Irvin, Myounghee Lee, Sailaja Kamaraju, Matthew Goetz, W. Fraser Symmans, Virginia Borges, Ian Krop, Ann Partridge, Lisa Carey. A011801 (CompassHER2 RD): Postneoadjuvant T-DM1 + tucatinib/placebo in patients with residual HER2-positive invasive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-18-11.

Abstract PO4-15-08: Comprehensive genomic profiling of HER2-low advanced breast cancers

Abstract Background: With the recent approval of the HER2-targeted antibody-drug conjugate (ADC) trastuzumab deruxtecan (T-Dxd) for the treatment of HER2-low advanced breast cancers (aBC), [defined as HER2 immunohistochemistry (IHC) 1+, or 2+ with negative in-situ hybridization (ISH)], there is interest to understand whether this population represents a distinct molecular entity and if there are additional biomarkers that can improve patient selection for these therapies. Here, we examined 2086 aBC cases profiled with comprehensive genomic profiling (CGP) stratified by HER2 status (negative, low, and positive) and hormone receptor (HR) status (positive and negative). Methods: Hybrid-capture CGP targeting 324 genes was performed on advanced BC samples (FoundationOne®CDx). HER2 IHC status was abstracted from pathology reports while HER2 amplification status was abstracted from CGP. Results: We examined 2086 samples of aBC patients distributed as HER2-negative (n=657; IHC 0), HER2-low (n=1012, IHC 1+, 2+ ISH non-amplified), and HER2-positive (n=417; HER2 IHC 3+). HER2-low patients were significantly older than HER2-negative patients (median 60 v 57 years, p=0.0006). HER2-low cases were more common in HR-positive than in HR-negative disease (67% vs 46%, respectively). Overall, characteristics were generally similar for genetic ancestry and TMB-H frequency across all HER2-statuses. MSI-H was rare across all HER2-statuses groups. HER2-low cases were more likely to have alterations in GATA3 (12.6% v 6.5%), ESR1 (12.4% v 7.8%), FGFR2 (4.0% v 1.8%), AR (1.1% v 0.2%), and CDH1 (15.3% v 11.6%) and less likely to harbor alterations in TP53 (44.0% v 62.4%), RB1 (6.8% v 12%), NF1 (5.5% v 9.3%), and BRCA1 (3.0% v 5.2%) when compared to HER2-negative. Some genes showed a consistent pattern of decreased frequency as HER2-IHC expression decreased from HER2-positive to HER2-low and to HER2-negative (e.g. AR alterations seen in 2.6%, 1.1% and 0.2%, respectively) while others had a increasing frequency pattern (e.g. RB1 alterations observed in 2.4%, 6.8% and 12.0%; BRCA1 seen in 1.7%, 3% and 5.2%, respectively). However, some genomic characteristics were unique to the HER2-low population (e.g. TP53 seen in 69.5% of HER2-positive and 62.4% of HER2-negative, but at a lower frequency of 44% in HER2-low cases) [Table 1]. Within the HR-positive subgroup, a lower frequency of NF1 (4.4% v 10.4%), TP53 (30.6% v 41.1%), and RB1 (4.4% v 8.0%) and a higher frequency of AR (1.0% v 0.0%) and GATA3 (16.2% v 10.9%) were observed in HER2-low vs HER2-negative cases, while within the HR-negative subgroup there was a higher frequency of GATA3 (1.2% v 0.7%) and AR (1.2% v 0.4%) and trending lower frequency of TP53 (85.7% v 90.8%) and RB1 (14.3% v 17.4%) in the HER2-low vs. HER2-negative cases. Mutations in homologous recombination repair genes (BRCA1, BRCA2, PALB2) were less frequent in HER2-low cases vs. HER2-negative cases overall (8.4% v 10.7%) and in the HR-positive (3.6% v 5.6%) and HR-negative subgroups (10.6% v 17.4%). Consistent with this, the rate of scar-based HRD signature (HRDsig) was lower in the HER2-low vs. HER2-negative subgroup (15.9% v 20.4%, p=0.02). Conclusions: These data suggest the HER2-low aBC has distinct genomic alterations before and after stratification by HR-status when compared to HER2-negative and HER2-positive aBC cases. Higher prevalence of some potentially actionable alterations in HER2-low cases, including FGFR2, AR, and ESR1, may aid in patient selection and drug development considering new combination approaches. Table 1 Summary of comprehensive genomic profiling alterations observed in HER2-low aBC compared to HER2-positive and HER2-negative cases Citation Format: Adriana Kahn, Ethan Sokol, Jeffrey Ross, Maureen Pelletier, Neal Fischbach, Lajos Pusztai, Maryam Lustberg. Comprehensive genomic profiling of HER2-low advanced breast cancers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-15-08.

Abstract CT168: A phase 1/2a, multicenter, open-label, non-randomized first-in-human study to assess the safety, tolerability, pharmacokinetics, and preliminary antitumor activity of DB-1310 (a HER3-targeting ADC) in patients with advanced/metastatic solid tumors

Abstract Background: Overexpression of epidermal growth factor receptor 3 (HER3 or ERBB3) has been reported in various cancers, including lung, prostate, head and neck, breast, and ovarian cancers, and is associated with disease progression and poor prognosis. DB-1310 is an antibody-drug conjugate (ADC) comprised of a humanized anti-HER3 IgG1 monoclonal antibody, covalently linked to a proprietary DNA topoisomerase I inhibitor (P1021) via a maleimide tetrapeptide-based cleavable linker, with a high drug-to-antibody ratio (~8). Preclinical studies of DB-1310 demonstrated promising antitumor activity in solid tumors and a tolerable safety profile, warranting further clinical development. Methods: This global, first-in-human, Phase 1/2a study includes dose escalation and expansion parts to assess the safety, tolerability, pharmacokinetics, and antitumor activity of DB-1310 in patients (pts) with pretreated advanced/metastatic solid tumors (NCT05785741). Eligible pts must have progressed on or after standard systemic anticancer treatments have ECOG PS ≤1, and evidence adequate organ function. Phase 1 part will evaluate 5 ascending dose levels of DB-1310 with a standard “3+3” design to identify the maximum tolerated dose (MTD) and/or recommended phase 2 dose (RP2D) in pts with advanced/metastatic solid tumors. Phase 2a part will initiate after MTD and/or RP2D are determined and enroll pts with non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) activating mutation, NSCLC without EGFR activating mutation, castration-resistant prostate cancer, head and neck squamous cell carcinoma, and HER2-positive (immunohistochemistry [IHC] 3+, or IHC 2+ and in situ hybridization-positive) breast cancer (BC). DB-1310 is administrated intravenously every 3 weeks as monotherapy, or plus trastuzumab in pts with HER2-positive BC only, until discontinuation criteria are met. The study plans to enroll approximately 95 pts in Phase 1 and 192 in Phase 2a from the United States and China. Citation Format: Aaron Lisberg, Shun Lu, Alexander Starodub, Harshad Amin, Julia Rotow, Lin Wu, Alexander Spira, Erika Hamilton, Weidi Shen, Liming Liu, Zhongyuan Zhu, Wei Gu, Yang Qiu, Rong Shi, Shengxue Liu. A phase 1/2a, multicenter, open-label, non-randomized first-in-human study to assess the safety, tolerability, pharmacokinetics, and preliminary antitumor activity of DB-1310 (a HER3-targeting ADC) in patients with advanced/metastatic solid tumors [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 CT168.

Abstract CT159: Phase I trial of trastuzumab deruxtecan in combination with neratinib in solid tumors with HER2 alterations (NCI 10495)

Abstract Background: Human epidermal growth factor receptor 2 (HER2) overexpression, amplification, or mutation occurs across a variety of solid tumors. Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate (ADC) that has been approved by the FDA for HER2-overexpressed advanced breast, gastroesophageal cancers, HER2-low breast cancer, and HER2-mutated non-small cell lung cancer. However, some patients do not derive clinical benefit from T-DXd, and drug resistance is inevitable. Preclinical studies demonstrated that neratinib, an oral irreversible pan-HER tyrosine kinase inhibitor, can increase uptake of HER2-targeting ADCs, including ado-trastuzumab emtansine and T-DXd, in vivo. Enhanced uptake of ADC is postulated to be secondary to hindering HSP90 binding to the HER2 receptor, thereby inducing receptor ubiquitination and internalization. This finding was further confirmed in patient-derived xenograft models from two patients with HER2-mutated, but non-amplified, low-expressing advanced breast cancer. Therefore, we hypothesized that the combination of T-DXd and neratinib in patients with advanced solid tumors with a HER2 alteration will enhance the efficacy of T-DXd. As T-DXd and neratinib may have overlapping toxicities in patients, a phase I study to establish the safety of the combination is warranted. Methods: This is a multi-center, phase I clinical trial (NCT05372614, NCI 10495) with a 3+3 design to study the safety and tolerability of T-DXd and neratinib. The trial is supported by the National Cancer Institute Experimental Therapeutics Clinical Trials Network. Eligible patients have advanced solid tumors with HER2 overexpression, amplification by ISH/NGS, or an activating HER2 mutation. Up to 18 evaluable patients will be enrolled in the dose escalation portion (Part 1), and 12 patients will be enrolled in the pharmacodynamic cohort (Part 2). During Part 1, the maximum tolerated dose and recommended phase 2 dose will be determined for neratinib. The dose of T-DXd is fixed at 5.4 mg/kg. We are actively enrolling patients in Part 1. The primary endpoint is dose-limiting toxicities of the combination during the first 2 cycles of treatment (42 days). Key secondary objectives of the study include T-DXd payload (DXd/MAAA-1181a) in tumor tissue obtained before and after co-treatment with neratinib to assess the potential enhanced payload concentration with the combination and quantitative HER2 assessed by mass spectrometry using multiplex reaction monitoring. Citation Format: Andrew A. Davis, Ecaterina E. Dumbrava, Aparna Kalyan, Nataliya V. Uboha, Haider S. Mahdi, Reema A. Patel, Farshid Dayyani, Jessica Porzel, Whitney L. Hensing, Cynthia X. Ma, Ron Bose, Haeseong Park. Phase I trial of trastuzumab deruxtecan in combination with neratinib in solid tumors with HER2 alterations (NCI 10495) [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 CT159.

Abstract 6006: Modeling antibody drug conjugate sensitivity using urothelial carcinoma patient-derived models

Abstract Introduction: Enfortumab Vedotin (EV), an antibody-drug conjugate (ADC) targeting Nectin-4, has emerged as a new standard-of-care for patients with metastatic urothelial cancer (UC). Promising results have also been observed in bladder cancer (BLCA) patients with ADCs targeting HER2. To identify predictive biomarkers of ADC sensitivity, we characterized a large and expanding biobank of patient-derived organoid (PDO) and xenograft (PDX) models for Nectin-4 and HER2 expression, and Nectin-4 and HER2-targeted ADC sensitivity. Methods: We generated 19 PDOs and 34 PDXs that reflect the genomic and biologic heterogeneity of human UC. A subset of these models was further characterized using a multiplatform approach, including bulk and single-cell DNA and RNA sequencing and immunoblot for Nectin-4 and HER2 expression. We leveraged these models to study the biologic determinants of sensitivity to EV and the HER2-targeted ADC trastuzumab deruxtecan (T-DXd). PDX models were treated with EV (5 mg/kg IV on day 1, 3, 6) or T-DXd (10 mg/kg IV on day 1, 21, 42) or vehicle only as control. We also studied sensitivity to T-DXd and EV, as well as to their cytotoxic payloads (exatecan and vedotin) in vitro using paired PDO models. Results: We observed high degree of variability of HER2 and Nectin-4 expression across UC PDOs and PDXs. In PDO models, sensitivity to EV correlated strongly with Nectin-4 expression, whereas HER2 expression was not strongly correlated with sensitivity to T-DXd. We also noted a poor correlation between Nectin-4 expression in PDOs and PDXs derived from the same patient. A subset of models, for example, SMBO-104 (ERBB2 wildtype with low HER2 expression), exhibited significantly greater sensitivity to EV than to T-DXd. Conversely, T-DXd was significantly more active than EV in SMBO-170, a model with ERBB2 amplification (~40 copies) and HER2 3+ expression. Sensitivity to exatecan, an analogue of the T-DXd payload deruxtecan, as measured by inhibition of cell proliferation and induction of apoptosis, was more predictive of T-DXd sensitivity than HER2 expression level. Conclusion: In this study, we generated and biologically characterized UC patient-derived models with the goal of identifying predictive biomarkers of sensitivity to Nectin-4 and HER2-targeted ADCs. We observed significant heterogeneity of HER2 and Nectin-4 expression across models. Models were identified in which T-DXd was superior to EV, and vice versa. Sensitivity to the T-DXd cytotoxic payload was more predictive of PDX response than HER2 expression levels, with significant tumor regression observed with T-DXd in BLCA models with low HER2 expression. Our data suggest that clinical trials of HER2-targeted ADCs such as T-DXd should not be restricted to patients with HER2 overexpression and that novel biomarkers will be needed to identify those UC patients most likely to benefit from FDA-approved and investigational ADCs. Citation Format: Xinran Tang, Ziyu Chen, Jasmine Thomas, Karan Nagar, John Christin, Naryan Rustgi, Sizhi Gao, Elisa de Stanchina, Jonathan Coleman, Michael Shen, Hikmat Al-Ahmadie, Gopa Iyer, Kwanghee Kim, David Solit. Modeling antibody drug conjugate sensitivity using urothelial carcinoma patient-derived 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 6006.

Abstract 6352: Preclinical discovery of ARX622: A site-specific, TLR7 agonist, HER2-targeted immune-stimulatory antibody drug conjugate for treatment of multiple solid tumor types

Abstract Traditional antibody-drug conjugates leverage the specificity of antibodies to deliver cytotoxic payloads to tumor microenvironments (TMEs), thereby reducing systemic toxicities associated with non-targeted chemotherapeutics. Analogously, a novel class of immune-stimulating antibody conjugates (ISACs) has recently emerged to selectively deliver synthetic immune agonists to the TME without systemic immune activation. The first generation of ISACs and the majority of approved and investigational ADCs contain payloads linked to native lysine or cysteine residues, which can lead to heterogenous drug-antibody ratios (DARs), reduced antibody stability, and labile conjugation, all of which limit therapeutic index. Here we describe the generation and preclinical characterization of ARX622, a site-specific TLR7-selective agonist ISAC targeting HER2. To avoid the instability issues of previous generation ADCs and ISACs, ARX622 has a homogenous and stable DAR via oxime bond-mediated payload conjugation to a synthetic amino acid, para-acetyl phenylalanine (pAF), which is recombinantly incorporated at defined heavy chain positions. Similar to cytotoxic ADCs built on this site-specific conjugation platform, ARX622 ISAC and total mAb pharmacokinetic (PK) profiles in non-human primates (NHPs) are indistinguishable, demonstrating in vivo stability and minimal payload deconjugation. Accordingly, ARX622 displays an extended PK profile in mice compared to a DAR-matched random cysteine ISAC containing the same antibody and payload. In the presence of HER2-expressing cells, ARX622 selectively induces proinflammatory cytokine production, type I and III IFN secretion, and APC maturation. In a syngeneic tumor model, ARX622 promotes complete tumor regressions, immunologic memory, and epitope spreading. In xenograft systems, single doses of ARX622 induce complete regression of large, established tumors (&amp;gt;1000mm3), and robustly inhibit tumor growth in mice that have progressed through prior treatment with HER2-targeted mAbs or ADCs. In HER2-low xenografts, ARX622 monotherapy inhibits tumor growth, and promotes complete regression of late-stage (800mm3) tumors when sequentially combined with a HER2-ADC. Based on PK data at efficacious doses in mice and tolerated exposures in NHP toxicity studies, ARX622 has an estimated therapeutic index of ~60x. Taken together, ARX622’s mAb-like properties, high stability, broad immune mechanism induction, strong anti-tumor activity alone or in combination with ADCs, and wide therapeutic index make it a strong candidate for further development toward treating patients with a variety of HER2-expressing solid tumors. Citation Format: David Mills, Ji Young Kim, Erica Wood, Keith Tatsukawa, Nick Knudsen, Jay Nelson, Lillian Skidmore, Kedar GC, Hon Tran, Manoj Pal, Hamidreza Hashemi, Ying Buechler, Shawn Zhang, Dan O'Connor. Preclinical discovery of ARX622: A site-specific, TLR7 agonist, HER2-targeted immune-stimulatory antibody drug conjugate for treatment of multiple solid tumor types [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 6352.

Abstract 6581: DX126-262 combined with chemotherapy (Cisplatin and 5-Fu) demonstrates promising antitumor efficacy in HER2-positive gastric cancer

Abstract Gastric cancer is a common malignant tumor of the digestive system, and its morbidity and mortality rank among the top five in the world. Although significant progress has been made in cytotoxic chemotherapy, immunotherapy and targeted therapy in recent years, the long-term prognosis of patients with advanced or metastatic gastric cancer is still poor. Trastuzumab combined with chemotherapy is the first-line standard of care (SOC) for HER2-positive advanced or metastatic gastric cancer. Two ADC drugs targeting HER2, DS-8201 and RC-48, have also been approved for the treatment of gastric cancer. However, their limited clinical benefits and occurrence of adverse events still present a significant challenge. Therefore, minimizing the off-target toxicity and collateral damage while achieving significant chemotherapy efficacy has become the focus of gastric cancer treatment. DX126-262 is a novel HER2-targeting antibody-drug conjugate (ADC), generated by conjugating a Tubulysin B analogue to a recombinant humanized anti-HER2 monoclonal antibody through a linker containing branched hydrophilic polyethylene glycol, using thiol-maleimide chemistry to achieve drug antibody ratio (DAR) of 3.8. Previous studies have confirmed that DX126-262 monotherapy showed excellent efficacy in HER2-positive gastric cancer NCI-N87 cells in vitro and in vivo. And the efficacy has also been demonstrated in clinical trials (now in phase II, CTR20211871). Based on above results, we intended to further explore whether combination therapy of DX126-262 with Cisplatin and 5-FU can improve the anti-tumor efficacy compared with SOC (Herceptin plus Cisplatin and 5-FU) or DS-8201 monotherapy. The triple-drug combination therapy demonstrated much better therapeutic efficacy than single drug (DX126-262, Cisplatin, 5-FU, Herceptin, or DS-8201) or double-drug combination (Cisplatin plus 5-FU) or SOC (Herceptin plus Cisplatin and 5-FU) on human gastric cancer cells in vitro and in vivo. Meanwhile, triple-drug combination therapy did not exhibit superimposed toxicity, judging by the body weight of mice and hemal biochemistry assays. These results suggested that DX126-262 plus Cisplatin and 5-FU might be a promising strategy for treatment of HER2-positive advanced or metastatic gastric cancer in clinic. Citation Format: Xiaobo Cai, Min Cao, Huihui Guo, Qingliang Yang, Xiangfei Kong, Yong Du, Zhicang Ye, Zhixiang Guo, Lingli Zhang, Lu Bai, Junxiang Jia, Yunxia Zheng, Yongxiang Chen, Miaomiao Chen, Wei Zheng, Jun Zheng, Wenjun Li, Yuanyuan Huang, Mengmeng Liu, Zhongliang Fan, Hangbo Ye, Yifang Xu, Binbin Chen, Meng Dai, Robert Y. Zhao. DX126-262 combined with chemotherapy (Cisplatin and 5-Fu) demonstrates promising antitumor efficacy in HER2-positive gastric cancer [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 6581.

Efficacy of Antibody Drug Conjugates Alone and in Combination with other Agents in Metastatic Urothelial Carcinoma: A Scoping Review.

Antibody drug conjugates represent a promising class of antineoplastic agents comprised of a monoclonal antibody linked to a potent cytotoxic payload for targeted delivery of chemotherapy to tumors. Various antibody drug conjugates have demonstrated impressive efficacy in patients with metastatic urothelial carcinoma in clinical trials, leading to two FDA approved therapies and several other agents and combinations in clinical development. A comprehensive systematic review was undertaken utilizing the principles of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. Queried databases included Ovid MEDLINE, Ovid Embase, Web of Science Core Collection and Cochrane CENTRAL Trials. The search sought to identify prospective therapeutic clinical trials in humans with metastatic urothelial carcinoma with a single-arm or randomized controlled trial design investigating antibody drug conjugate-containing regimens. The literature search yielded 4,929 non-duplicated articles, of which 30 manuscripts and conference abstracts were included, which derived from 15 clinical trials including 19 separate cohorts with efficacy outcome results. Eleven trials investigated ADC monotherapy, while two investigated combination regimens, and the remaining two studies were mixed. Five unique ADC targets were represented including Nectin-4, Trop-2, HER2, Tissue Factor, and SLITRK6. Twelve clinical trial cohorts required prior treatment (63%). Objective response rate was reported for all studies and ranged from 27-52% for ADC monotherapies and 34-75% for ADC plus anti-PD-1 agents. Time to event outcome reporting was highly variable. In addition to enfortumab vedotin and sacituzumab govitecan, various HER2-targeted antibody drug conjugates and ADC-anti-PD-1 combination regimens have demonstrated efficacy in clinical trials and are poised for clinical advancement.

Elucidating molecular mechanisms and therapeutic synergy: irreversible HER2-TKI plus T-Dxd for enhanced anti-HER2 treatment of gastric cancer

BackgroundHER2-targeted therapies have improved the outcomes of HER2-positive gastric cancer (GC), yet resistance remains a challenge. We sought to explore the effects of reversible and irreversible HER2 tyrosine kinase inhibitors (TKIs) alone or in combination with the HER2-targeting antibody drug conjugate trastuzumab deruxtecan (T-Dxd).MethodsThe effects of HER2-TKIs on HER2 and downstream signaling were evaluated via Western blotting. Proteasomal inhibitors and co-immunoprecipitation assays were performed to explore the role of proteasomal degradation in HER2 expression modulation, and immunofluorescence assays were employed to explore mechanisms of HER2 internalization. The synergistic potential of the irreversible HER2-TKI pyrotinib in combination with T-Dxd was validated using growth and viability assays in anti-HER2-positive GC cell cultures and tumor growth and immunohistochemical staining assays in a mouse xenograft model.ResultsOur study revealed that reversible HER2-TKIs elevated HER2 protein levels, whereas irreversible HER2-TKIs decreased them. Pyrotinib triggered HER2 degradation within the proteasome by promoting ubiquitination and dissociation from HSP90. Furthermore, pyrotinib substantially induced HER2 internalization, which led to improved cellular uptake of T-Dxd. The increased T-Dxd uptake was accompanied by greater efficacy in suppressing the growth of GC cells and enhanced anti-tumor effects in an animal model.ConclusionIn summary, our research reveals the molecular mechanisms of irreversible HER2-TKIs in regulating HER2 protein expression by promoting HER2 internalization. These findings advance our comprehension of targeted therapy for GC and provide a promising therapeutic combination strategy with enhanced efficacy against HER2-positive GC.