Inhibition Of Epidermal Growth Factor Receptor Signaling Research Articles

Abstract LB056: IBI334, a novel ADCC-enhanced B7-H3/EGFR bispecific antibody, demonstrated potent pre-clinical efficacy in solid tumors

Abstract Many solid tumors, such as colorectal cancer (CRC) and lung cancer, are dependent on epidermal growth factor receptor (EGFR) pathway for tumor growth. Antibodies that inhibit EGFR signaling, such as Cetuximab, show clinical efficacy but are associated with on-target toxicities that potentially limit the drug efficacy. To enhance the EGFR signaling inhibition and reduce on-target toxicities, we performed bioinformatics analysis for EGFR/TAA co-expression. We found that B7-H3, a member of the B7 family of immune checkpoint proteins, is broadly expressed in many solid tumors and their expression correlates with EGFR expression in multiple cancers, including lung cancer, head and neck cancer, pancreatic cancer and esophageal cancer. Using Innovent’s proprietary Innobody platform, we developed a bispecific antibody, IBI334, against B7-H3 and EGFR. IBI334 has an EGFR arm for signal blockage and is coupled with a fine-tuned B7-H3 arm with optimal affinity and binding domain. With the aid of B7-H3, IBI334 showed enhanced EGFR signal inhibition than Zalutumumab (EGFR mAb) and Amivantamab (c-met/EGFR bsAb). In addition, IBI334 is afucosylated to enhance its antibody-mediated cell cytotoxicity (ADCC) effects as compared to wild type antibody. IBI334 showed significantly better in vivo efficacy than Amivantamab in NCI-H358 (bronchioalveolar carcinoma with KRAS-G12C mutation), NCI-H292 (lymph node metastasis of a pulmonary mucoepidermoid carcinoma), SK-MES1 (lung cancer) and SK-MES1-LTC (lung cancer with EGFR L858R, T790M and C797S mutations) xenograft models and comparable efficacy in NCI-H322-EGFRexon20ins (lung cancer) model. Cetuximab, an EGFR monoclonal antibody, had high on-target toxicity with 50% early mortalities in cynomolgus monkeys at 120/75 mg/kg/week. With the careful design of B7-H3/EGFR bispecific antibody, IBI334 ameliorated the on-target toxicities and showed only slight hardening of the skin at the injection site at 120 mg/kg/week. As a result, IBI334 has a large therapeutic window of >200 folds. In conclusion, IBI334 is a potent B7-H3/EGFR bispecific antibody with broad application in many EGFR-driven solid tumors. The large therapeutic window enables safe and effective cancer treatment at high exposure levels. Citation Format: Jian Guan, Shuaixiang Zhou, Weiwei Wu, Tianyu Zhu, Lei Cao, Ming Wu, Ninghuan Li, Fenggen Fu, Zhengguan Liao, Shuming Lin, Zeyu Liu, Mengjia Zhu, Nana Luo, Ying Zhang, Tiong Sun Chia, Bingliang Chen, Kaijie He. IBI334, a novel ADCC-enhanced B7-H3/EGFR bispecific antibody, demonstrated potent pre-clinical efficacy in 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 LB056.

Abstract 1866: Discovery of mutation-independent EGFR degrading bispecific antibodies that suppress tumor growth in preclinical tumor models

Abstract Extracellular targeted protein degradation (eTPD) has emerged as a promising new drug modality focused on targeted elimination of extracellular and transmembrane proteins. In contrast to intracellular protein degraders, such as proteolysis targeting chimeras (PROTACs) and molecular glues which require ubiquitin-proteosome cellular degradation pathways, extracellular protein degraders can additionally harness endosomal-lysosomal protein degradation. Two recently published examples of extracellular protein degraders include AbTACs (antibody-based PROTAC) which co-engage a protein of interest (POI) and transmembrane E3 ligases, and KineTACs (cytokine receptor-targeting chimeras) which utilize endogenous cytokine receptors to degrade extracellular POIs. These bispecific antibody degrader platforms not only have advantageous pharmacological and drug-like manufacturing properties, but can also be engineered for tissue-specificity and to address multiple complementary targets, with the goal of increased efficacy and decreased toxicity. To that end, we have greatly expanded the extracellular degrader repertoire beyond AbTACs and KineTACs, with a novel bispecific antibody degrader platform called TrainTACs (tissue receptor antigen internalization targeting chimeras). To demonstrate the potential of this novel extracellular degrading platform, we developed degraders for the canonical receptor tyrosine kinase epidermal growth factor receptor (EGFR). EGFR is an oncogenic driver, that has been clinically validated in lung, colorectal and head and neck cancers, but patient benefit has been limited by treatment-related acquired resistance mutations and on-target/off-tumor dose-limiting toxicities. The potential of our TrainTACs, AbTACs and KineTACs to overcome the limitations of current therapeutic modalities was assessed. Gene expression profiles from tumor and normal tissues were evaluated to identify potential degraders co-expressed with EGFR in tumors. More than 70 unique bispecific antibody constructs spanning 20 receptors were generated and screened in tumor cell-based assays to evaluate EGFR antagonism, internalization, and degradation. TrainTACs, AbTACs and KineTACs degraded EGFR in multiple tumor cell lines covering a range of EGFR mutations. Degradation of EGFR led to deep inhibition of EGFR signaling, robust inhibitory effects on tumor spheroids, and in xenograft mouse tumor models. In conclusion, eTPD represents a promising new drug modality, and TrainTACs, AbTACs and KineTACs have expanded the toolbox of extracellular targeted protein degraders that can be utilized in a target-, tissue- and disease-specific manner. Citation Format: Jonathan Sitrin, Lisa Marshall, Hai Tran, Kenneth Ng, Kimberly Hoi, Josef Gramespacher, Zhong Huang, Andy Goodrich, Filomena Housley, May Dayao, Man-Tzu Wang, Katarina Pance, Aleysha Chen, Kevin Carlin, Lichao Zhang, James Lee, Rami Hannoush, Ken Flanagan, Maia Vinogradova, Isaac Rondon, Shyra Gardai. Discovery of mutation-independent EGFR degrading bispecific antibodies that suppress tumor growth in preclinical tumor 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 1866.

HB-EGF promotes progenitor cell proliferation and sensory neuron regeneration in the zebrafish olfactory epithelium.

Maintenance and regeneration of the zebrafish olfactory epithelium (OE) are supported by two distinct progenitor cell populations that occupy spatially discrete stem cell niches and respond to different tissue conditions. Globose basal cells (GBCs) reside at the inner and peripheral margins of the sensory OE and are constitutively active to replace sporadically dying olfactory sensory neurons (OSNs). In contrast, horizontal basal cells (HBCs) are uniformly distributed across the sensory tissue and are selectively activated by acute injury conditions. Here we show that expression of the heparin-binding epidermal growth factor-like growth factor (HB-EGF) is strongly and transiently upregulated in response to OE injury and signals through the EGF receptor (EGFR), which is expressed by HBCs. Exogenous stimulation of the OE with recombinant HB-EGF promotes HBC expansion and OSN neurogenesis in a pattern that resembles the tissue response to injury. In contrast, pharmacological inhibition of HB-EGF membrane shedding, HB-EGF availability, and EGFR signaling strongly attenuate or delay injury-induced HBC activity and OSN restoration without affecting maintenance neurogenesis by GBCs. Thus, HB-EGF/EGFR signaling appears to be a critical component of the signaling network that controls HBC activity and, consequently, repair neurogenesis in the zebrafish OE.

Abstract C036: A Phase 1 Study to assess BDTX-1535, an oral EGFR inhibitor, in patients with Glioblastoma or Non-Small Cell Lung Cancer

Abstract Background: Epidermal growth factor receptor (EGFR) is a potent oncogene commonly altered in non-small cell lung cancer (NSCLC) and glioblastoma (rGBM). For NSCLC, EGFR tyrosine kinase activity can be driven by classical driver, intrinsic driver, and acquired resistance mutations. BDTX-1535 is an orally available, highly potent, brain penetrant, selective, irreversible small molecule tyrosine kinase inhibitor that targets all 3 classes of EGFR mutations. Preclinical data demonstrated the ability of BDTX-1535 to cross the blood-brain barrier and produce sustained inhibition of EGFR signaling. Methods: BDTX-1535-101 (NCT05256290) is a Phase 1, open-label, multicenter study to assess the safety, tolerability, pharmacokinetics (PK), central nervous system (CNS) activity, and preliminary antitumor activity of BDTX-1535 in locally advanced or metastatic NSCLC with or without CNS disease or recurrent GBM (rGBM). The primary objective is to determine the BDTX-1535 recommended Phase 2 dose (RP2D) based on the overall safety, PK, pharmacodynamic, and preliminary antitumor activity. The monotherapy dose escalation portion evaluates BDTX-1535 in patients with locally advanced/metastatic NSCLC harboring intrinsic driver EGFR mutations following standard of care or with acquired resistance EGFR mutations following a 3rd generation EGFR inhibitor in the 1st line setting, in addition to patients with rGBM expressing EGFR alterations who previously received available standard therapy of surgical resection followed by chemoradiotherapy and/or temozolomide (TMZ). A capsule-to-tablet bridging cohort and a food effect cohort will also be assessed. Following the establishment of a preliminary recommended Phase 2 dose (RP2D), the disease-specific expansion portion of the study will further evaluate the safety, tolerability, and preliminary antitumor effect of BDTX-1535. Cohorts include the following:1) advanced/metastatic NSCLC with intrinsic resistance EGFR mutations following up to 2 lines of therapy, one of which must be an EGFR inhibitor treatment, 2) advanced/metastatic NSCLC with acquired resistance EGFR mutation following a 3rd generation EGFR inhibitor in the first line setting, 3) rGBM with confirmed EGFR alterations with or without amplifications. NSCLC patients may enroll with or without CNS metastases and must not be known to express excluded resistance mutations such as EGFR T790M. BDTX-1535 will also be studied in combination with TMZ to assess safety, tolerability, and a recommended combination dose for the treatment of patients with newly diagnosed GBM harboring EGFR alterations. Enrollment was initiated in 2022 and dose escalation is almost complete. Initiation of the dose expansion cohorts of NSCLC patients with EGFR acquired resistance and intrinsic driver mutations after progression on prior EGFR inhibitor is planned for the second half of 2023. For additional information, please contact ClinicalTrials@bdtx.com. Citation Format: Helena Yu, Melissa Johnson, Jason T Henry, Alex Spira, Ji-Youn Han, Minal Barve, James Battiste, Iyad Alnahhas, DoHyun Nam, Jeffrey Edenfield, Balazs Halmos, Shinkyo Yoon, Tae Min Kim, Sudharshan Eathiraj, Julio Hajdenberg, Sergey Yurasov, Manmeet Ahluwalia, Patrick Wen. A Phase 1 Study to assess BDTX-1535, an oral EGFR inhibitor, in patients with Glioblastoma or Non-Small Cell Lung Cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C036.

LHPP suppresses proliferation, migration, and invasion in hepatocellular carcinoma and pancreatic cancer by inhibiting EGFR signaling pathway.

Phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) has been reported to be a new tumor suppressor with a significant inhibitory effect in various cancers. Although LHPP has been repeatedly shown to inhibit the progression of various tumors by inhibiting the phosphorylation of AKT, up to now, the studies on the function and mechanism of LHPP in tumors are insufficient. In this study, LHPP expression was found to be downregulated in both hepatocellular carcinoma (HCC) and pancreatic cancer (PC). Here, we found that LHPP could bind to epidermal growth factor receptor (EGFR) and inhibit its phosphorylation, which thereby inhibited the activation of EGFR downstream pathways ERK, AKT, and STAT3, and then weakening the ability to proliferate, invade, and migrate in HCC and PC. This paper showed a new physiological function of LHPP in inhibiting phosphorylation of EGFR and its potential anti-tumor mechanism and indicated that LHPP was a potential therapeutic target for HCC and PC.

Tubular cell transcriptional intermediary factor 1γ deficiency exacerbates kidney injury-induced tubular cell polyploidy and fibrosis

Tubulointerstitial fibrosis is considered the final convergent pathway of progressive chronic kidney diseases (CKD) regardless of etiology. However, mechanisms underlying kidney injury-induced fibrosis largely remain unknown. Recent studies have indicated that transcriptional intermediary factor 1γ (TIF1γ) inhibits the progression of fibrosis in other organs. Here, we found that TIF1γ was highly expressed in the cytoplasm and nucleus of the kidney proximal tubule. Interestingly, we found tubular TIF1γ expression was decreased in patients with CKD, including those with diabetes, hypertension, and IgA nephropathy, and in mouse models with experimental kidney fibrosis (unilateral ureteral obstruction [UUO], folic acid nephropathy [FAN], and aristolochic acid-induced nephrotoxicity). Tubule-specific knock out of TIF1γ in mice exacerbated UUO- and FAN-induced tubular cell polyploidy and subsequent fibrosis, whereas overexpression of kidney TIF1γ protected mice against kidney fibrosis. Mechanistically, in tubular epithelial cells, TIF1γ exerted an antifibrotic role via transforming growth factor-β (TGF-β)-dependent and -independent signaling. TIF1γ hindered TGF-β signaling directly by inhibiting the formation and activity of the transcription factor Smad complex in tubular cells, and we discovered that TIF1γ suppressed epidermal growth factor receptor (EGFR) signaling upstream of TGF-β signaling in tubular cells by ubiquitylating EGFR at its lysine 851/905 sites thereby promoting EGFR internalization and lysosomal degradation. Pharmacological inhibition of EGFR signaling attenuated exacerbated polyploidization and the fibrotic phenotype in mice with tubule deletion of TIF1γ. Thus, tubular TIF1γ plays an important role in kidney fibrosis by suppressing profibrotic EGFR and TGF-β signaling. Hence, our findings suggest that maintaining homeostasis of tubular TIF1γ may be a new therapeutic option for treating tubulointerstitial fibrosis and subsequent CKD.

Hydrogen peroxide-induced oxidative damage and protective role of peroxiredoxin 6 protein via EGFR/ERK signaling pathway in RPE cells.

Damage to retinal pigment epithelium (RPE) cells caused by oxidative stress is closely related to the pathogenesis of several blinding retinal diseases, such as age-related macular degeneration (AMD), retinitis pigmentosa, and other inherited retinal degenerative conditions. However, the mechanisms of this process are poorly understood. Hence, the goal of this study was to investigate hydrogen peroxide (H2O2)-induced oxidative damage and protective role of peroxiredoxin 6 (PRDX6) protein via EGFR/ERK signaling pathway in RPE cells. Cells from a human RPE cell line (ARPE-19 cells) were treated with H2O2, and then cell viability was assessed using the methyl thiazolyl tetrazolium assay. Cell death and reactive oxygen species (ROS) were detected by flow cytometry. The levels of PRDX6, epidermal growth factor receptor (EGFR), P38 mitogen-activated protein kinase (P38MAPK), c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) were detected by Western blot assay. PRDX6 and EGFR were also detected via immunofluorescence staining. Our results show that H2O2 inhibited cell viability, induced cell death, and increased ROS levels in ARPE-19 cells. It was also found that H2O2 decreased the levels of PRDX6, EGFR, and phosphorylated ERK but increased the levels of phosphorylated P38MAPK and JNK. PRDX6 overexpression was found to attenuate H2O2-induced inhibition of cell viability and increased cell death and ROS production in ARPE-19 cells. PRDX6 overexpression also increased the expression of EGFR and alleviated the H2O2-induced decrease in EGFR and phosphorylated ERK. Moreover, inhibition of epidermal growth factor-induced EGFR and ERK signaling in oxidative stress was partially blocked by PRDX6 overexpression. Our findings indicate that PRDX6 overexpression protects RPE cells from oxidative stress damage caused by decreasing ROS production and partially blocking the inhibition of the EGFR/ERK signaling pathway induced by oxidative stress. Therefore, PRDX6 shows promise as a therapeutic target for the prevention of RPE cell damage caused by oxidative stress associated with retinal diseases.

Inhibition of VEGFR2 and EGFR signaling cooperatively suppresses the proliferation of oral squamous cell carcinoma.

Epidermal growth factor receptor (EGFR) is frequently overexpressed in oral squamous cell carcinoma (OSCC), and EGFR-targeting therapeutics have been widely employed to treat patients with a variety of carcinomas including OSCC. Here, we aimed to investigate alternative signaling for OSCC survival under the disruption of EGFR signaling. OSCC cell lines, namely HSC-3 and SAS, were utilized to investigate how EGFR disruption affects cell proliferation. Gene set enrichment analysis was performed to examine how EGFR disruption affects oncogenic signaling in OSCC cells. Disruption of KDR gene was performed using CRISPR/Cas9 techniques. A VEGFR inhibitor, vatalanib was used to research the impact of VEGFR inhibition on OSCC survival. EGFR disruption significantly decreased the proliferation and oncogenic signaling including Myc and PI3K-Akt, in OSCC cells. Chemical library screening assays revealed that VEGFR inhibitors continued to inhibit the proliferation of EGFR-deficient OSCC cells. In addition, CRISPR-mediated disruption of KDR/VEGFR2 retarded OSCC cell proliferation. Furthermore, combined erlotinib-vatalanib treatment exhibited a more potent anti-proliferative effect on OSCC cells, compared to either monotherapy. The combined therapy effectively suppressed the phosphorylation levels of Akt but not p44/42. VEGFR-mediated signaling would be an alternative signaling pathway for the survival of OSCC cells under the disruption of EGFR signaling. These results highlight the clinical application of VEGFR inhibitors in the development of multi-molecular-targeted therapeutics against OSCC.

A phase 1 study to assess BDTX-1535, an oral EGFR inhibitor, in patients with glioblastoma or non–small-cell lung cancer.

TPS9156 Background: The epidermal growth factor receptor (EGFR) is a potent oncogene commonly altered in many cancers, including glioblastoma (GBM) and non-small cell lung cancer (NSCLC). EGFR tyrosine kinase activity driven by common EGFR mutations can be inhibited by small molecules, however, resistance to available agents may be driven by mutations in the EGFR active kinase site or other regions. BDTX-1535 is an orally available, highly potent, selective, irreversible inhibitor of EGFR mutations, including extracellular variants and amplifications commonly expressed in GBM and inhibits the common and uncommon EGFR mutations found in NSCLC, including the C797S mutation acquired following 3rd generation EGFR inhibitor therapy. Preclinical data demonstrated the ability of BDTX-1535 to cross the blood-brain barrier and produce sustained inhibition of EGFR signaling. Preclinical studies suggest that BDTX-1535 has the potential to be clinically active in suppressing tumor growth in patients with GBM and NSCLC with or without CNS metastases, including a potential survival benefit. Methods: BDTX-1535-101 (NCT05256290) is Phase 1, open-label, multicenter study to assess the safety, tolerability, PK, CNS activity, and preliminary antitumor activity of BDTX-1535 in recurrent GBM (rGBM) or locally advanced or metastatic NSCLC with or without CNS disease. The Monotherapy Dose Escalation portion will evaluate BDTX-1535 in patients with either rGBM expressing EGFR alterations or locally advanced/metastatic NSCLC harboring sensitizing EGFR mutations with or without CNS disease. Patients with rGBM must have previously received available standard therapy of surgical resection followed by chemoradiotherapy and/or temozolomide (TMZ). Eligible NSCLC patients must have EGFR mutated NSCLC that has progressed following standard of care EGFR inhibitor therapy. Once a provisional recommended Phase 2 dose (RP2D) has been established, BDTX-1535 monotherapy will be explored in the following Dose Expansion cohorts to further evaluate safety, PK, and preliminary assessment of efficacy: 1) rGBM with confirmed EGFR alterations, 2) NSCLC with uncommon EGFR mutations following EGFR inhibitor therapy; 3) NSCLC with acquired EGFR resistance mutation following a 3rd generation EGFR inhibitor in 1L setting. NSCLC patients may enroll with or without CNS metastases and must not be known to express excluded resistance mutations such as EGFR T790M or MET. BDTX-1535 will also be studied in combination with TMZ to assess safety, tolerability, and a recommended combination dose for the treatment of patients with rGBM harboring EGFR mutations or variants. Enrollment was initiated in 2022 and dose escalation is ongoing. Dose Expansion cohorts are expected to open in 2023. For additional information, please contact BDTX_1535_101_Study@bdtx.com . Clinical trial information: NCT05256290 .

Abstract CT127: A phase 1 study to assess BDTX-1535, an oral EGFR inhibitor, in patients with glioblastoma or non-small cell lung cancer

Abstract Background: The epidermal growth factor receptor (EGFR) is a potent oncogene commonly altered in many cancers, including glioblastoma (GBM) and non-small cell lung cancer (NSCLC). EGFR tyrosine kinase activity driven by common EGFR mutations can be inhibited by small molecules, however, resistance to available agents may be driven by mutations in the EGFR active kinase site or other regions. BDTX-1535 is an orally available, highly potent, selective, irreversible inhibitor of EGFR mutations, including extracellular variants and amplifications commonly expressed in GBM and inhibits the uncommon EGFR mutations found in NSCLC, including the C797S mutation acquired following 3rd generation EGFR inhibitor therapy. Preclinical data demonstrated the ability of BDTX-1535 to cross the blood-brain barrier and produce sustained inhibition of EGFR signaling. Preclinical studies suggest that BDTX-1535 has potential to be clinically active in suppressing tumor growth in patients with GBM and NSCLC with or without CNS metastases, including a potential survival benefit. Methods: BDTX-1535-101 (NCT05256290) is Phase 1, open-label, multicenter study to assess the safety, tolerability, PK, CNS penetrance, and preliminary antitumor activity of BDTX-1535 in recurrent GBM (rGBM) or locally advanced or metastatic NSCLC with or without CNS disease. The Monotherapy Dose Escalation portion will evaluate BDTX-1535 in patients with either rGBM expressing EGFR alterations or locally advanced/metastatic NSCLC harboring sensitizing EGFR mutations with or without CNS disease. Patients with rGBM must have previously received available standard therapy of surgical resection followed by chemoradiotherapy and/or temozolomide (TMZ). Eligible NSCLC patients must have EGFR mutated NSCLC that has progressed following standard of care EGFR inhibitor therapy. Once a provisional recommended Phase 2 dose (RP2D) has been established, BDTX-1535 monotherapy will be explored in the following Dose Expansion cohorts to further evaluate safety, PK, and preliminary assessment of efficacy: 1) rGBM with confirmed EGFR alterations, 2) NSCLC with uncommon EGFR mutations following EGFR inhibitor therapy; 3) NSCLC with acquired EGFR resistance mutation following a 3rd generation EGFR inhibitor in 1L setting. NSCLC patients may enroll with or without CNS metastases and must not be known to express excluded resistance mutations such as EGFR T790M or MET. BDTX-1535 will also be studied in combination with TMZ to assess safety, tolerability, and a recommended combination dose for the treatment of patients with rGBM harboring EGFR mutations or variants. Enrollment was initiated in 2022 and dose escalation is ongoing. Dose Expansion cohorts are expected to open in 2023. For additional information, please contact BDTX_1535_101_Study@bdtx.com Citation Format: Melissa Johnson, Jason Henry, Alex Spira, James Battiste, Iyad Alnahhas, Manmeet Ahluwalia, Minal Barve, Jeffrey Edenfield, DoHyun Nam, Sudharshan Eathiraj, Julio Hajdenberg, Sergey Yurasov, Helena Yu, Patrick Wen. A phase 1 study to assess BDTX-1535, an oral EGFR inhibitor, in patients with glioblastoma or non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT127.

Deletion of DYRK1A Accelerates Osteoarthritis Progression Through Suppression of EGFR-ERK Signaling.

Dual-specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) signaling is involved in the dynamic balance of catabolism and anabolism in articular chondrocytes. This study aimed to investigate the roles and mechanism of DYRK1A in the pathogenesis of osteoarthritis (OA). The expressions of DYRK1A and its downstream signal epidermal growth factor receptor (EGFR) were detected in the cartilage of adult wild-type mice with destabilized medial meniscus (DMM) and articular cartilage of patients with OA. We measured the progression of osteoarthritis in chondrocyte-specific knockout DYRK1A(DYRK1A-cKO) mice after DMM surgery. Knee cartilage was histologically scored and assessed the effects of DYRK1A deletion on chondrocyte catabolism and anabolism. The effect of inhibiting EGFR signaling in chondrocytes from DYRK1A-cKO mice was analyzed. Trauma-induced OA mice and OA patients showed downregulation of DYRK1A and EGFR signaling pathways. Conditional DYRK1A deletion aggravates DMM-induced cartilage degeneration, reduces the thickness of the superficial cartilage, and increases the number of hypertrophic chondrocytes. The expression of collagen type II, p-ERK, and aggrecan was also downregulated, and the expression of collagen type X was upregulated in the articular cartilage of these mice. Our findings suggest that DYRK1A delays the progression of knee osteoarthritis in mice, at least in part, by maintaining EGFR-ERK signaling in articular chondrocytes.

Abstract 6319: Mechanism of anti-EGFR antibody endostatin fusion protein action on inhibition of vasculogenic mimicry and tumor cell motility in triple negative breast cancer

Abstract Triple Negative Breast Cancer (TNBC) is an aggressive subtype of breast cancer with high metastatic potential and increased morbidity and mortality. Although expression of and signaling by the epidermal growth factor receptor (EGFR) is commonly seen in TNBC, anti-EGFR antibodies such as Cetuximab have had limited therapeutic efficacy, used alone or in combination with chemotherapy. Primary TNBC tumor growth and metastases require supporting vasculature, which develops through a combination of endothelial angiogenesis and vasculogenic mimicry (VM). VM is frequently seen in TNBC and is associated with aggressive metastatic behavior. We previously developed αEGFR-E-P125A, an antibody-endostatin fusion protein, linking an anti-EGFR antibody to a mutated version of the anti-angiogenic protein endostatin (E-P125A). αEGFR-E-P125A delivers a dimeric E-P125A payload which inhibits TNBC angiogenesis and VM in vitro and in vivo, and markedly decreases metastasis in multiple TNBC xenograft models. To study changes in the transcriptome following induction of VM and inhibition of VM with αEGFR-E-P125A, RNA-seq was conducted on MDA-MB-231-4175 TNBC cells grown in a 2D monolayer and compared to TNBC cells undergoing VM when plated in matrigel. A second comparison was made between TNBC cells undergoing VM and TNBC cells in which VM was inhibited by treatment with αEGFR-E-P125A. Differential gene expression analysis and gene set enrichment analysis (GSEA), were used to define pathways which were inversely regulated during the induction of VM on matrigel and inhibition of VM with αEGFR-E-P125A. Gene set enrichment analysis demonstrated that αEGFR-E-P125A treatment significantly downregulated genes in the JAK-STAT and angiogenesis signaling pathways. Phospho-array analysis demonstrated decreased EGFR phosphorylation at Y1069 in addition to decreased phosphorylation of FAK Y397 and STAT3 Y705 sites downstream of α5β1 integrin. Since inhibition of EGFR signaling alone did not inhibit VM, and endostatin is a known ligand of α5β1 integrin, we focused on effects of αEGFR-E-P125A on inhibition of α5β1 integrin/FAK signaling pathway. Independent siRNA knockdowns of α5 integrin and FAK in MDA-MB-231-4175 cells confirmed that downregulation of α5 integrin/FAK signaling inhibited VM in vitro. αEGFR-E-P125A additionally prevented EGF-induced EGFR phosphorylation and fibronectin-induced FAK phosphorylation by α5β1 integrin. Treatment of TNBC cells with αEGFR-E-P125A reduced total α5 integrin protein levels and decreased co-localization of EGFR and α5β1 integrin receptors. These results indicate that αEGFR-E-P125A suppressed both EGFR and α5β1 integrin signaling. Simultaneous inhibition of EGFR and α5β1integrin signaling by αEGFR-E-P125A fusion is a promising approach to inhibition of TNBC growth and metastases. Citation Format: Ankita Sankar, Hava Gil Henn, Hyun Mi Cho, Dania Nassar, Sundaram Ramakrishnan, Yu Zhang, Christian Elledge, Seung Uon Shin, Joseph Rosenblatt. Mechanism of anti-EGFR antibody endostatin fusion protein action on inhibition of vasculogenic mimicry and tumor cell motility in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6319.

Abstract 4999: Preclinical evaluation of MCLA-129, a bispecific antibody targeting EGFR and c-MET on solid tumor cells, in comparison with amivantamab

Abstract The HGF/c-MET pathway is frequently upregulated in tumors that are resistant to EGFR tyrosine kinase inhibitors. MCLA-129 is an antibody-dependent cellular cytotoxicity (ADCC) enhanced Biclonics® that targets epidermal growth factor receptor (EGFR) and c-MET in non-small cell lung cancer (NSCLC) and other solid tumors. MCLA-129 is a potent inhibitor of tumor growth applying various mechanisms of action (MOA), including inhibition of c-MET and EGFR signaling, antibody-dependent cellular phagocytosis (ADCP) and ADCC. Here we compared the MOA of MCLA-129 with the MOA of the EGFR-c-MET bispecific antibody amivantamab. The binding of MCLA-129 to tumor cells expressing different levels of EGFR and c-MET was quantified using radiolabeled antibodies. Scatchard plot analysis showed that the affinity of MCLA-129 for both c-MET and EGFR is in the low nanomolar range. In FACS, MCLA-129 and amivantamab show similar binding to tumor cells harboring EGFR or c-MET exon14-skipping mutations. Crossblock experiments with the c-MET binding antibody onartuzumab analog showed that MCLA-129 competes for the same HGF-binding region of c-MET, which is distinct from the binding site of amivantamab according to Neijssen et al, 2021 (PMC8113745). MCLA-129 and amivantamab did not downmodulate EGFR or c-MET. ADCP was also comparable for MCLA-129 and amivantamab, as measured with monocyte-derived macrophages incubated with pHrodo-labeled target cells with readout of tumor cell phagocytosis on the Incucyte® system. In contrast, MCLA-129 displayed ADCC activity similar to or more potently than amivantamab in an ADCC reporter assay with either high affinity FcγRIII 158V-variant or low affinity 158F-variant effector cells in absence or presence of soluble EGFR and soluble c-MET. MCLA-129-induced ADCC appears to be less affected than amivantimab by the presence of soluble EGFR and soluble c-MET. In conclusion, MCLA-129 is a Biclonics® common light chain bispecific antibody with multiple MOA including inhibition of c-MET and EGFR ligand binding, ADCP and ADCC comparable or more potent than amivantamab. A phase 1/2 clinical trial of MCLA-129 in solid tumors is ongoing. These data support the further clinical development of MCLA-129 in patients with NSCLC and other solid tumors. Citation Format: David J.J. de Gorter, Alexandre Deshiere, Arjen Kramer, Martijn van Rosmalen, Franziska Mortensen, Cecile Geuijen. Preclinical evaluation of MCLA-129, a bispecific antibody targeting EGFR and c-MET on solid tumor cells, in comparison with amivantamab. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4999.

Abstract P2-24-06: αEGFR-E-P125A ANTIBODY-ENDOSTATIN FUSION PROTEIN REDUCES VASCULOGENIC MIMICRY AND TUMOR CELL MOTILITY BY INHIBITION OF EGFR, INTEGRIN AND FAK SIGNALING

Abstract Triple Negative Breast Cancer (TNBC) is an aggressive subtype of breast cancer with high metastatic potential and increased morbidity and mortality. Although expression of, and signaling by the epidermal growth factor receptor (EGFR) is commonly seen in TNBC, anti-EGFR antibodies such as Cetuximab have had limited therapeutic efficacy, used alone or in combination with chemotherapy. Primary TNBC tumor growth and metastases require supporting vasculature, which develops through a combination of endothelial angiogenesis and vasculogenic mimicry (VM). VM is more frequently seen in TNBC than other breast cancer subtypes, and is associated with aggressive metastatic behavior. We previously developed αEGFR-E-P125A, an antibody-endostatin fusion protein, linking an anti-EGFR antibody targeting domain to a mutated version of the anti-angiogenic protein endostatin (E-P125A). αEGFR-E-P125A delivers a dimeric E-P125A payload which inhibits TNBC angiogenesis and VM in vitro and in vivo, and markedly decreases metastasis in both the MDA-MB-231-4175 and MDA-MB-468 TNBC xenograft models. To determine the mechanism of αEGFR-E-P125A action on inhibition of VM, we studied the effects of αEGFR-E-P125A on both the transcriptome and proteome. RNA-seq was conducted on MDA-MB-231-4175 TNBC cells plated on matrigel and undergoing VM, and on TNBC cells plated in matrigel and treated with αEGFR-E-P125A. Gene set enrichment analysis demonstrated that αEGFR-E-P125A treatment downregulated genes on the KRAS, JAK-STAT, and angiogenesis signaling pathways, including EGF, VEGFA, STAT3, PTK2, ITGB1, ITGB3, ITGAV, and ITGA5. Phospho-array analysis was used to interrogate the proteome and demonstrated downregulation of phosphorylation on multiple sites downstream of the EGFR and the α5β1 integrin receptors, such as the EGFR Y1069 site, the FAK Y397 site, implicated in the regulation of endothelial and TNBC motility, and the STAT3 Y705 and S727 sites, known for their role in promoting the transcription of angiogenic genes. Since inhibition of EGFR signaling alone does not inhibit VM, we interrogated the mechanistic relationship between αEGFR-E-P125A and VM inhibition through the α5β1 integrin/FAK signaling pathway. Specific inhibition of FAK at the Y397 site using the small molecule inhibitor, PF-573228 inhibited TNBC VM in vitro. Transient siRNA knockdown of FAK in MDA-MB-231-4175 cells and shRNA-mediated knockdown of FAK in MDA-MB-231 TNBC cells confirmed that downregulation of FAK inhibited VM in vitro. To understand the effect of αEGFR-E-P125A on the EGFR and α5β1 integrin receptors, competition assays were conducted. αEGFR-E-P125A competed with EGF for the EGFR receptor and with the binding of fibronectin to α5β1 integrin. Treatment of TNBC cells with αEGFR-E-P125A reduced total α5 integrin protein levels and decreased co-localization of EGFR and α5β1 integrin receptors. These results indicate that αEGFR-E-P125A is bound to both EGFR and α5β1 integrin, simultaneously suppressing downstream EGFR and integrin signaling. Simultaneous inhibition of EGFR and α5β1integrin signaling by αEGFR-E-P125A fusion is a promising approach to inhibition of TNBC growth and metastases. Citation Format: Ankita P. Sankar, Hava Gil Henn, Hyun Mi Cho, Dania Nassar, Sundaram Ramakrishnan, Rathin Das, Yu Zhang, Christian Elledge, Seung-Uon Shin, Joseph Rosenblatt. αEGFR-E-P125A ANTIBODY-ENDOSTATIN FUSION PROTEIN REDUCES VASCULOGENIC MIMICRY AND TUMOR CELL MOTILITY BY INHIBITION OF EGFR, INTEGRIN AND FAK SIGNALING [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-24-06.

Bruceine D and afatinib combination inhibits ovarian cancer cells proliferation and migration through DNA damage repair and EGFR pathway.

Owing to the high rates of relapse and migration, ovarian cancer (OC) has been recognized as the most lethal gynecological malignancy worldwide. The activity of the epidermal growth factor receptor (EGFR) signaling pathway is frequently associated with OC cell proliferation and migration. Despite this knowledge, inhibition of EGFR signaling in OC patients failed to achieve satisfactory therapeutic effects. In this study, we identified that bruceine D (BD) and EGFR inhibitor, afatinib, combination resulted in synergistic anti-OC effects. The results indicated that compared with one of both drugs alone, the combination of BD and afatinib slowed the DNA replication rate, inhibition of cell viability, and proliferation and clone formation. This resulted in cell cycle arrest and cell apoptosis. In addition, the combination of BD and afatinib possessed a stronger ability to inhibit the OC cell adhesion and migration than treatment with BD or afatinib alone. Mechanistically, the combined treatment triggered intense DNA damage, suppressed DNA damage repair, and enhanced the inhibition of the EGFR pathway. These results demonstrated that compared with each pathway inhibition, combined blocking of both DNA damage repair and the EGFR pathway appears to more effective against OC treatment. The results support the potential of BD and afatinib combination as a therapeutic strategy for OC patients.

Recombinant Human Prolidase (rhPEPD) Induces Wound Healing in Experimental Model of Inflammation through Activation of EGFR Signalling in Fibroblasts

The potential of recombinant human prolidase (rhPEPD) to induce wound healing in an experimental model of IL-1β-induced inflammation in human fibroblasts was studied. It was found that rhPEPD significantly increased cell proliferation and viability, as well as the expression of the epidermal growth factor receptor (EGFR) and downstream signaling proteins, such as phosphorylated PI3K, AKT, and mTOR, in the studied model. Moreover, rhPEPD upregulated the expression of the β1 integrin receptor and its downstream signaling proteins, such as p-FAK, Grb2 and p-ERK 1/2. The inhibition of EGFR signaling by gefitinib abolished rhPEPD-dependent functions in an experimental model of inflammation. Subsequent studies showed that rhPEPD augmented collagen biosynthesis in IL-1β-treated fibroblasts as well as in a wound healing model (wound closure/scratch test). Although IL-1β treatment of fibroblasts increased cell migration, rhPEPD significantly enhanced this process. This effect was accompanied by an increase in the activity of MMP-2 and MMP-9, suggesting extracellular matrix (ECM) remodeling during the inflammatory process. The data suggest that rhPEPD may play an important role in EGFR-dependent cell growth in an experimental model of inflammation in human fibroblasts, and this knowledge may be useful for further approaches to the treatment of abnormalities of wound healing and other skin diseases.

WSD-0922, a novel brain-penetrant inhibitor of epidermal growth factor receptor, promotes survival in glioblastoma mouse models.

Although the epidermal growth factor receptor (EGFR) is a frequent oncogenic driver in glioblastoma (GBM), efforts to therapeutically target this protein have been largely unsuccessful. The present preclinical study evaluated the novel EGFR inhibitor WSD-0922. We employed flank and orthotopic patient-derived xenograft models to characterize WSD-0922 and compare its efficacy to erlotinib, a potent EGFR inhibitor that failed to provide benefit for GBM patients. We performed long-term survival studies and collected short-term tumor, plasma, and whole-brain samples from mice treated with each drug. We utilized mass spectrometry to measure drug concentrations and spatial distribution and to assess the impact of each drug on receptor activity and cellular signaling networks. WSD-0922 inhibited EGFR signaling as effectively as erlotinib in in vitro and in vivo models. While WSD-0922 was more CNS penetrant than erlotinib in terms of total concentration, comparable concentrations of both drugs were measured at the tumor site in orthotopic models, and the concentration of free WSD-0922 in the brain was significantly less than the concentration of free erlotinib. WSD-0922 treatment provided a clear survival advantage compared to erlotinib in the GBM39 model, with marked suppression of tumor growth and most mice surviving until the end of the study. WSD-0922 treatment preferentially inhibited phosphorylation of several proteins, including those associated with EGFR inhibitor resistance and cell metabolism. WSD-0922 is a highly potent inhibitor of EGFR in GBM, and warrants further evaluation in clinical studies.

A supramolecular self-assembled nanomaterial for synergistic therapy of immunosuppressive tumor

Triple negative breast cancer (TNBC) is an immunosuppressive "cold" tumor that lacks immune cell infiltration and activation, resulting in a poor response to immune checkpoint blockade (ICB) therapies. In addition, TNBC is poorly responsive to targeted therapies due to the absence of efficient molecular targets. A strategy that can block molecular signal transduction, stimulate immunogenicity, and activate the immune response is a promising approach to achieve ideal clinical benefit. Herein, we designed and synthesized an aggregation-induced emission luminogen (AIEgen)-conjugated self-assembling peptide that targets epidermal growth factor receptor (EGFR), named TPA-FFG-LA. TPA-FFG-LA peptides form nanoassemblies on the surface of EGFR-positive TNBC cells and are internalized into cells through endocytosis, which inhibit EGFR signaling transduction and provoke lysosomal membrane permeabilization (LMP). Upon light irradiation, the aggregated AIEgens produce massive reactive oxygen species (ROS) to exacerbate LMP and trigger immunogenic cell death (ICD), resulting in elimination of residual EGFR-negative tumor cells and exerting long-term antitumor effects. The in vitro and in vivo experiments verified that TPA-FFG-LA nanoassemblies suppress tumor growth, provoke immune cell activation and infiltration, and cause EGFR degradation and LMP. These results suggest that the combination of supramolecular assembly induced molecular targeting effects and lysosome dysfunction with ICD-stimulated immune activation is a plausible strategy for the efficient therapy of immunosuppressive TNBC.

Dual Inhibition of EGFR and IGF-1R Signaling Leads to Enhanced Antitumor Efficacy against Esophageal Squamous Cancer.

Both the epidermal growth factor receptor (EGFR) and insulin-like growth factor 1 receptor (IGF-1R) have been implicated in the development of cancers, and the increased expression of both receptors has been observed in esophageal cancer. However, the tyrosine kinase inhibitors of both receptors have thus far failed to provide clinical benefits for esophageal cancer patients. Studies have confirmed the complicated crosstalks that exist between the EGFR and IGF-1R pathways. The EGFR and IGF-1R signals act as mutual compensation pathways, thereby conveying resistance to EGFR or IGF-1R inhibitors when used alone. This study evaluated the antitumor efficacy of the EGFR/HER2 inhibitors, gefitinib and lapatinib, in combination with the IGF-1R inhibitor, linsitinib, on the esophageal squamous cell carcinoma (ESCC). Gefitinib or lapatinib, in combination with linsitinib, synergistically inhibited the proliferation, migration, and invasion of ESCC cells, caused significant cell cycle arrest, and induced marked cell apoptosis. Their combination demonstrated stronger inhibition on the activation of EGFR, HER2, and IGF-1R as well as the downstream signaling molecules. In vivo, the addition of linsitinib to gefitinib or lapatinib also potentiated the inhibition effects on the growth of xenografts. Our results suggest the next clinical exploration of the combination of gefitinib or lapatinib with linsitinib in the treatment of ESCC patients.

Pharmacological and In Silico Analysis of Oat Avenanthramides as EGFR Inhibitors: Effects on EGF-Induced Lung Cancer Cell Growth and Migration.

Avena sativa L. is a wholegrain cereal and an important edible crop. Oats possesses high nutritional and health promoting values and contains high levels of bioactive compounds, including a group of phenolic amides, named avenanthramides (Avns), exerting antioxidant, anti-inflammatory, and anticancer activities. Epidermal growth factor receptor (EGFR) represents one of the most known oncogenes and it is frequently up-regulated or mutated in human cancers. The oncogenic effects of EGFR include enhanced cell growth, angiogenesis, and metastasis, and down-regulation or inhibition of EGFR signaling has therapeutic benefit. Front-line EGFR tyrosine kinase inhibitor therapy is the standard therapy for patients with EGFR-mutated lung cancer. However, the clinical effects of EGFR inhibition may be lost after a few months of treatment due to the onset of resistance. Here, we showed the anticancer activity of Avns, focusing on EGFR activation and signaling pathway. Lung cancer cellular models have been used to evaluate the activity of Avns on tumor growth, migration, EMT, and anoikis induced by EGF. In addition, docking and molecular dynamics simulations showed that the Avns bind with high affinity to a region in the vicinity of αC-helix and the DGF motif of EGFR, jeopardizing the target biological function. Altogether, our results reveal a new pharmacological activity of Avns as EGFR tyrosine kinase inhibitors.