Targeting Epidermal Growth Factor Receptor Research Articles

Betacellulin regulates gap junction intercellular communication by inducing the phosphorylation of connexin 43 in human granulosa-lutein cells

BackgroundThe gap junction protein, connexin 43 (Cx43) is highly expressed in human granulosa-lutein (hGL) cells. The phosphorylation of certain amino acid residues in the Cx43 protein has been shown to be related to a decline in gap junction intercellular communication (GJIC), which subsequently affects oocyte meiotic resumption. As a member of the epidermal growth factor (EGF) family, betacellulin (BTC) mediates luteinizing hormone (LH)-induced oocyte maturation and cumulus cell expansion in mammalian follicles. Whether BTC can regulate Cx43 phosphorylation, which further reduces Cx43-coupled GJIC activity in hGL cells remains to be determined.MethodsImmortalized human granulosa cells (SVOG cells) and primary human granulosa-lutein cells obtained from women undergoing in vitro fertilization in an academic research center were used as the study models. The expression levels of Cx43 and phosphorylated Cx43 were examined following cell incubation with BTC at different time points. Several kinase inhibitors (sotrastaurin, AG1478, and U0126) and small interfering RNAs targeting EGF receptor (EGFR) and receptor tyrosine-protein kinase 4 (ErbB4) were used to verify the specificity of the effects and to investigate the molecular mechanisms. Real-time-quantitative PCR and western blot analysis were used to detect the specific mRNA and protein levels, respectively. GJIC between SVOG cells were evaluated using a scrape loading and dye transfer assay. Results were analyzed by one-way analysis of variance.ResultsThe results showed that BTC induced the rapid phosphorylation of Cx43 at serine368 without altering the expression of Cx43 in primary and immortalized hGL cells. Additionally, using a dual inhibition approach (kinase inhibitors and siRNA-based expression knockdown), we demonstrated that this effect was mainly mediated by the EGFR but not the ErbB4 receptor. Furthermore, using a protein kinase C (PKC) kinase assay and a scrape-loading and dye transfer assay, we revealed that PKC signaling is the downstream signaling pathway that mediates the increase in Cx43 phosphorylation and subsequent decrease in GJIC activity in response to BTC treatment in hGL cells.ConclusionsBTC promptly induced the phosphorylation of connexin 43 at Ser368, leading to decreased GJIC activity in hGL cells. The BTC-induced cellular activities were most likely driven by the EGFR-mediated PKC-dependent signaling pathway. Our findings shed light on the detailed molecular mechanisms by which BTC regulates the process of oocyte meiotic resumption.

A Novel and Highly Selective Epidermal Growth Factor Receptor Inhibitor, SMUZ106, for the Treatment of Glioblastoma.

Targeting the epidermal growth factor receptor (EGFR) is one of the potential ways to treat glioblastoma (GBM). In this study, we investigate the anti-GBM tumor effects of the EGFR inhibitor SMUZ106 in both in vitro and in vivo conditions. The effects of SMUZ106 on the growth and proliferation of GBM cells were explored through MTT and clone formation experiments. Additionally, flow cytometry experiments were conducted to study the effects of SMUZ106 on the cell cycle and apoptosis of GBM cells. The inhibitory activity and selectivity of SMUZ106 to the EGFR protein were proved by Western blotting, molecular docking, and kinase spectrum screening methods. We also conducted a pharmacokinetic analysis of SMUZ106 hydrochloride following i.v. or p.o. administration to mice and assessed the acute toxicity level of SMUZ106 hydrochloride following p.o. administration to mice. Subcutaneous and orthotopic xenograft models of U87MG-EGFRvIII cells were established to assess the antitumor activity of SMUZ106 hydrochloride in vivo. SMUZ106 could inhibit the growth and proliferation of GBM cells, especially for the U87MG-EGFRvIII cells with a mean IC50 value of 4.36 μM. Western blotting analyses showed that compound SMUZ106 inhibits the level of EGFR phosphorylation in GBM cells. It was also shown that SMUZ106 targets EGFR and presents high selectivity. In vivo, the absolute bioavailability of SMUZ106 hydrochloride was 51.97%, and its LD50 exceeded 5000 mg/kg. SMUZ106 hydrochloride significantly inhibited GBM growth in vivo. Furthermore, SMUZ106 inhibited the activity of U87MG-resistant cells induced by temozolomide (TMZ) (IC50: 7.86 μM). These results suggest that SMUZ106 hydrochloride has the potential to be used as a treatment method for GBM as an EGFR inhibitor.

Exploration of Indolo[3,2c]isoquinoline derived triazoles as potential antimicrobial and DNA cleavage agents: Synthesis, DFT calculations, and molecular modeling studies

Indole and its derivatives are well-known assorted motif in drug design and development. We here in reporting synthesis of new 9-chloro-1-(4-substituted phenyl)-12H-indolo[2,3-c][1,2,4]triazolo[3,4-a]isoquinolines 7 (a-h). Structures of the newly synthesized compounds were confirmed by making use of spectroscopic techniques like IR, NMR and Mass. The DFT calculations were taken for the selected molecules using CAM-B3LYP hybrid functional with a 6–31 + g(d) all-electron basis set using the Gaussian 09 package. The drug-likeness predictions were described for the synthesized derivatives. The In vitro antimicrobial and DNA cleavage activities were reported for all compounds 7 (a-h). The compounds 7a, 7b, and 7h showed excellent microbial inhibition and DNA cleavage activity as compared to standard drugs. Furthermore, the docking studies for the newly synthesized molecules were carried out by Auto dock software with two molecular targets Epidermal Growth Factor Receptor tyrosine kinase (1 M17) and C-kit Tyrosine Kinase (1 T46) exhibited better binding affinity of all synthesized compounds. In addition, the docking results were observed to be in full agreement with the in vitro DNA cleavage assay suggesting the potential of synthesized metal complexes in biological applications. Lastly, the protein stability, fluctuations of APO-Protein, and protein–ligand complexes were investigated through Molecular Dynamics (MD) simulations studies using Desmond Maestro 11.3 and potential lead molecules were identified.

Molecular Docking and Simulation-Binding Analysis of Plant Phytochemicals with the Hepatocellular Carcinoma Targets Epidermal Growth Factor Receptor and Caspase-9.

Among primary liver cancers, hepatocellular carcinoma (HCC) is one of the most common forms and it has been categorized as the joint-fourth largest reason of cancer-related deaths globally. Different factors such as alcohol abuse, hepatitis B and C, viral infections, and fatty liver diseases are mainly related to the pathogenesis of HCC. In the current study, 1000 total various plant phytochemicals were docked to proteins involved in HCC. The compounds were docked to the active site amino acids of epidermal growth factor receptor and caspase-9 as receptor proteins in order to explore their inhibiting potential. The top five compounds against each receptor protein were explored as potential drug candidates on the basis of their binding affinity and root-mean square deviation values. The top two compounds against each protein were found to be liquoric acid (S-score -9.8 kcal/mol) and madecassic acid (S-score -9.3 kcal/mol) against EGFR, and limonin (S-score -10.5 kcal/mol) and obamegine (S-score -9.3 kcal/mol) against the caspase-9 protein. The selected phytochemicals were further assessed through drug scanning using Lipinski's rule of five to explore their molecular properties and druggability. According to the ADMET analysis, the selected phytochemicals were found to be non-toxic and non-carcinogenic. Finally, the molecular dynamics simulation study revealed that liquoric acid and limonin were stabilized within the binding pockets of EGFR and capase-9, respectively, and stayed firmly bound throughout the simulation. In light of the current findings, the phytochemicals reported in this study, especially liquoric acid and limonin, could be used as potential drugs for the treatment of HCC in the future.

Abstract CT156: MCLA-158 (petosemtamab), an IgG1 bispecific antibody targeting EGFR and LGR5, in advanced gastric/esophageal adenocarcinoma (GEA)

Abstract The epidermal growth factor receptor (EGFR) is a known driver of cancer growth and the leucine-rich, repeat-containing, G-protein coupled receptor 5 (LGR5) is a transmembrane receptor expressed on cancer stem cells. Petosemtamab is a human, common light chain, IgG1 bispecific antibody with enhanced ADCC activity targeting EGFR and LGR5, which has shown potent antitumor activity in patient (pt)-derived xenograft models of gastric and esophageal cancer. The dose escalation part of an ongoing phase 1/2 study is completed (JCO 2021:39.3 Sup 62). In the expansion part, petosemtamab is being investigated at the RP2D (1500 mg Q2W, 4-week cycle) in pts with selected advanced solid tumors, including GEA. Primary objective (expansion): investigator-assessed ORR per RECIST 1.1. Secondary objectives: ORR, DOR, PFS (per investigator and central review), OS, and safety and tolerability. Key eligibility criteria: advanced/metastatic GEA, prior exposure to ≥2 lines of standard therapy, ECOG PS 0-1, measurable disease (RECIST 1.1), available baseline tumor biopsy. At the data cutoff date of 24 October 2022, 14 GEA pts were treated at the RP2D. Median age was 63 years (range 40-80), ECOG PS 0/1: 3/11 pts, and 79% of pts were male. Primary tumor locations were esophagus (57%), stomach (36%), and gastroesophageal junction (7%), and all were adenocarcinoma histology. Pts received a median of 3 lines of prior systemic therapy (range 1-4) including platinum-based chemotherapy (100%) and checkpoint inhibitors (14%). 3/14 pts had EGFR overexpression (H score ≥200). A median of 2 treatment cycles (range 1-24) was administered, with 1 pt continuing therapy at the cutoff. Among the 14 pts evaluable for efficacy (≥2 cycles and ≥1 postbaseline scan, or early progression) limited activity was seen, however 1 pt with tumor EGFR protein overexpression and gene copy number amplification (CNA) showed a confirmed sustained PR (67% tumor reduction; response ongoing after 24 cycles) and 3 pts had SD (1 with EGFR overexpression and gene CNA; 2 not evaluable for IHC), with tumor reductions of 2%, 17%, and 40%. Among 78 pts treated at the RP2D (escalation and all expansion cohorts; cutoff 28 November 2022), the most frequent AEs regardless of causality (all grades/G3-4) were rash (33%/0%), hypotension (26%/6%), dyspnea (26%/4%), nausea (26%/1%), dermatitis acneiform (24%/1%), blood magnesium decreased (19%/5%), erythema (19%/0%), diarrhea (19%/0%); IRRs (composite term) were reported in 74%/21% of pts, mostly at the first infusion, and all resolved. 5 pts (6%) discontinued treatment due to IRRs on Day 1. 1 esophageal cancer pt died due to unrelated G5 GI bleeding. Petosemtamab demonstrated promising clinical efficacy among patients with pretreated GEA having EGFR gene amplification and/or overexpression, with a manageable safety profile. Citation Format: Marc Díez García, Antoine Hollebecque, Rocio Garcia-Carbonero, Christiane Jungels, Elisabeth Smyth, Shumei Kato, Guillem Argilés, Carlos Gomez Martin, Marina Magin, Yu-Ming Shen, Renée de Leeuw, Mohamed Bekradda, Eduardo Pennella, Ernesto Wasserman, Viktoriya Stalbovskaya, Jeroen Lammerts van Bueren, Aline Engbers, Andrew Joe, Josep Tabernero. MCLA-158 (petosemtamab), an IgG1 bispecific antibody targeting EGFR and LGR5, in advanced gastric/esophageal adenocarcinoma (GEA) [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 CT156.

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.

Changes in T lymphocyte subsets in peripheral blood of patients with middle-advanced cervical cancer before and after nimotuzumab combined with concurrent chemoradiotherapy

The current study sought to investigate the effect of nimotuzumab combined with concurrent chemoradiotherapy (CCRT + Nim) on T lymphocyte subsets in middle-advanced CC. Firstly, patients with middle-advanced CC were administered CCRT or CCRT + Nim. Next, levels of T lymphocytes in peripheral blood of CC patients pre- or post-treatment and healthy females were determined by flow cytometry. The short-term efficacy was evaluated, and overall survival (OS) and progression-free survival (PFS) of patients were recorded. In addition, the correlation of T lymphocyte subsets post-treatment with OS/PFS was assessed with Pearson analysis. CC patients exhibited decreased total T cells/T helper cells/CD4+/CD8+ ratio and increased T suppressor cells/Tregs in peripheral blood. Meanwhile, CCRT and CCRT + Nim improved T lymphocyte subset imbalance, with CCRT + Nim exhibiting better efficacy. CCRT + Nim exhibited better short-term efficacy and higher PFS than CCRT, with no evident difference in OS. The levels of total T cells/T helper cells/T suppressor cells/Tregs were not significantly-correlated with OS/PFS, and the CD4+/CD8+ ratio was correlated with PFS but not OS. Collectively, CCRT + nimotuzumab ameliorate the imbalance of T lymphocyte subsets in peripheral blood of middle-advanced CC patients, and the CD4+/CD8+ ratio after therapy is correlated with PFS. IMPACT STATEMENT What is already known on this subject? The utilisation of Nimotuzumab targeting epidermal growth factor receptor (EGFR) combined with concurrent chemoradiotherapy (CCRT) as an efficient treatment for middle-advanced cervical cancer (CC) has garnered the attention of numerous researchers over the years. T cells represent a major immune cell type in the tumour microenvironment and serve as the basis for maintaining cellular immune functions. What do the results of this study add? Our findings revealed that nimotuzumab combined with CCRT improves the abnormality of T lymphocyte subsets in peripheral blood of patients with middle-advanced CC, such that the CD4+/CD8+ ratio after treatment was significantly correlated with progression-free survival (PFS). What are the implications of these findings for clinical practice and/or further research? CCRT of CC may have a short-term negative impact on the peripheral T-cell immune micro-environment, and the combination of nimotuzumab, cisplatin-based chemotherapy, and radiotherapy enhances the frequency of Tregs in peripheral blood. Our findings illustrated that nimotuzumab combined with CCRT can improve the imbalance of T lymphocyte subsets in peripheral blood of patients with middle-advanced CC. A better understanding of the mechanisms of these therapies will optimise the selection of patients most likely to benefit from treatment, serving as a reference for further research on the relationship between EGFR-specific T cells and clinical benefit in patients treated with nimotuzumab in combination with CCRT.

Biochemical separation of Cetuximab-Fab from papain-digested antibody fragments and radiolabeling with 64Cu for potential use in radioimmunotheranostics

Engineered Fab fragments of monoclonal antibodies (mAbs) after radiolabeling with suitable radiometals have the potential to play a key role in personalized radioimmunotheranostics of cancer patients. In this study, we have generated Fab fragment of Cetuximab, a mAb targeting epidermal growth factor receptor (EGFR) expression and purified from the Fc and other fragments by ultrafiltration and affinity chromatography. The Cetuximab-Fab was conjugated with a suitable bifunctional chelator and radiolabeled with no-carrier-added (NCA) 64Cu produced via 64Zn (n, p) 64Cu reaction in a nuclear reactor. The radioimmunoconjugate obtained after size exclusion chromatographic separation possessed >95% radiochemical purity and it retained its integrity over at least three half-lives of the radiometal. Biodistribution studies was performed in fibrosarcoma tumor bearing Swiss mice, which demonstrated the explicit need for purification of the Cetuximab-Fab from Fc fragments. Enhanced and rapid tumor uptake with decent tumor-to-background ratio with prolonged retention was observed when radiolabeled purified Cetuximab-Fab was intravenously administered in animal models. Overall, this preclinical study established the pivotal role of separation science and technology to obtain the radioimmunoconjugate with requisite purity in order to demonstrate optimal pharmacokinetics and maximized treatment efficacy.

EGFR is a potential therapeutic target for highly glycosylated and aggressive pancreatic neuroendocrine neoplasms.

pNENs are relative indolent tumors with heterogeneous clinical presentation at diagnosis. It is important to establish aggressive subgroups of pNENs and identify potential therapeutic targets. Patients with pNEN (322 cases) were included to examine the association between glycosylation biomarkers and clinical/pathological traits. The molecular and metabolic features stratified by glycosylation status were assessed by RNA-seq/whole exome sequencing and immunohistochemistry. A considerable proportion of patients had elevated glycosylation biomarkers (carbohydrate antigen [CA] 19-9, 11.9%; CA125, 7.5%; carcinoembryonic antigen [CEA], 12.8%). CA19-9 (hazard ratio [HR]=2.26, P=.019), CA125 (HR=3.79, P=.004) and CEA (HR=3.16, P=.002) were each independent prognostic variables for overall survival. High glycosylation group, defined as pNENs with elevated level of circulating CA19-9, CA125 or CEA, accounted for 23.4% of all pNENs. High glycosylation (HR=3.14, P=.001) was an independent prognostic variable for overall survival and correlated with G3 grade (P < .001), poor differentiation (P=.001), perineural invasion (P=.004) and distant metastasis (P < .001). Epidermal growth factor receptor (EGFR) was enriched in high glycosylation pNENs using RNA-seq. EGFR was expressed in 21.2% of pNENs using immunohistochemistry and associated with poor overall survival (P=.020). A clinical trial focusing on EGFR expressed pNENs was initiated (NCT05316480). Thus, pNEN with aberrant glycosylation correlates with a dismal outcome and suggests potential therapeutic target of EGFR.

The NLRP3 Inflammasome Works as a Sensor for Detecting Hypoactivity of the Mitochondrial Src Family Kinases.

Gefitinib (GF), the tyrosine kinase inhibitor (TKI) targeting epidermal growth factor receptor, initiates lung inflammation through the NLR family pyrin domain containing 3 (NLRP3) inflammasome. However, the molecular targets and mechanisms underlying the inflammatory action of GF remain unknown. In this study, we identified mitochondrial Src family kinases (mSFKs) as key determinants of GF-induced NLRP3 inflammasome activation. Comprehensive analysis of the TKIs revealed that all TKIs we tested act as potent agonists for the NLRP3 inflammasome in human monocytic THP-1 cells and bone marrow-derived macrophages. Moreover, these TKIs share a common off-target activity against the mSFKs, such as c-Src, Fgr, and Fyn. Interestingly, loss of each kinase spontaneously stimulated the NLRP3 inflammasome activation in THP-1 cells. These results together suggest that NLRP3 senses hypoactivity of the mSFKs that is responsible for mitochondrial dysfunction. Thus, our findings demonstrate a mechanistic link between the NLRP3 inflammasome and mSFKs, which, to our knowledge, provides insights into a novel molecular basis and cellular function of the NLRP3 inflammasome.

Abstract OT2-14-01: Pyrotinib after trastuzumab-based adjuvant therapy in high-risk early human epidermal growth factor receptor 2-positive breast cancer: a multicenter phase 2 trial

Abstract Objective: Trastuzumab-based adjuvant therapy was demonstrated to improve the prognosis of early human epidermal growth factor receptor 2 (HER-2) positive breast cancer, while about 10% of patients showed primary resistance. ExteNET study showed that 1 year of neratinib, an irreversible pan-HER tyrosine kinase inhibitor, after trastuzumab-based adjuvant therapy significantly improved invasive disease-free survival (iDFS) in women with HER2-positive breast cancer. Besides, for patients with early hormone receptor positive breast cancer within 1 year of prior trastuzumab with residual disease after neoadjuvant therapy, extended adjuvant neratinib showed prolonged overall survival, which suggested the potential benefit of extended adjuvant therapy for high-risk patients. Pyrotinib is an oral irreversible pan-HER receptor tyrosine kinase inhibitor targeting epidermal growth factor receptor, HER2, and HER4, which showed promising efficacy and manageable safety profiles in the treatment of HER-2 positive advanced breast cancer. In this study, we aimed to evaluate the efficacy and safety of extended adjuvant pyrotinib following trastuzumab-based adjuvant therapy in patients with high-risk early HER-2 positive breast cancer. Methods: In this ongoing multicenter, open-label, phase II trial at 23 centers in China, women with operable high-risk early HER-2 positive breast cancer and known hormone receptor status, who have completed trastuzumab-based adjuvant therapy within 6 months were enrolled. High-risk patients must meet one of the following criteria: N stage ≥1; T stage ≥2; did not achieve pathological complete response (pCR) after neoadjuvant therapy; had pCR after neoadjuvant therapy but with tumor size ≥ 5cm or N stage ≥2; or tumor size less than 2cm but with high Ki67, histologic grade 3 or with lymph node micrometastasis. Eligible patients were assigned to receive 6 month or 1 year of oral pyrotinib 400mg/day. The primary endpoint was the 2-year iDFS rate. Results: Between January, 2019 and February, 2022, a total of 142 eligible women were enrolled and assigned to receive pyrotinib, with a median age of 50 (range: 25-72) years old. Ninety-seven patients had a T stage ≥2. A total of 108 patients (76.1%) had node-positive disease, and 64 (45.1%) were hormone receptor positive. Twenty-five patients used neoadjuvant therapy previously, and all of them did not receive pCR (Table 1). After a median follow-up of 20 (range: 0.25-41) months, the 2-year iDFS rate was not available. Two and four patients reported invasive disease events after 6-month and 1-year of follow-up, with the 6-month and 1-year iDFS rates of 98.6% and 97.2%, respectively. The most common adverse events reported was diarrhea (78.2%), followed by fatigue (36.6%), lymphocyte count decreased (36.6%), nausea (33.1%) and hand-foot syndrome (33.1%). Forty-three patients experienced grade 3 diarrhea, and no grade 4 or higher adverse event was reported (Table 2). Adverse event leading to treatment discontinuation occurred in 12 (8.5%) patients. Conclusion: Extended adjuvant pyrotinib administrated after trastuzumab-based adjuvant therapy was well-tolerated and showed potential efficacy in high-risk early HER-2 positive breast cancer. The follow-up is ongoing to determine the long-term benefit of extended adjuvant pyrotinib. Table 1. Baseline characteristics of patients ER: estrogen receptor; PR: progesterone receptor. Table 2. Treatment-emergent adverse event occurring in at least 10% of the patients Citation Format: Feilin Cao, Zhaosheng Ma, Guinv Hu, Xiaotao Zhu, Shuguang Li, Shuzheng Chen, Binglie Chen, Zhanwen Li, Weizhu Wu, Xiaochun Ji, Jingde Shu, Deyou Tao, Xiaoqing Hu, Min Zheng, Ouchen Wang, Qingjing Feng, Jing Hao, Xujun Li. Pyrotinib after trastuzumab-based adjuvant therapy in high-risk early human epidermal growth factor receptor 2-positive breast cancer: a multicenter phase 2 trial [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 OT2-14-01.

Revisiting targeted therapy and immunotherapy for advanced cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a rare and aggressive type of malignant tumor. In the past few years, there has been an increase in the incidence of CCA. Surgery is the only effective treatment but is only suitable for a small percentage of patients. Comprehensive treatment is the normal therapy for terminal CCA patients, depending basically on gemcitabine and cisplatin combination chemotherapy. In the past decade, the emergence of next-generation sequencing technology can be used for the identification of important molecular features of CCA, and several studies have demonstrated that different CCA subtypes have unique genetic aberrations. Targeting fibroblast growth factor receptor (FGFR), isocitrate dehydrogenase (IDH) and epidermal growth factor receptor 2 (EGFR2) are emerging targeted therapies. In addition, researches have indicated that immunotherapy has a key function in CCA. There is ongoing research on programmed cell death protein 1 inhibitors (PD-1), chimeric antigen receptor T cells (CAR-T) and tumor-infiltrating leukocyte (TILs). Researches have shown that targeted therapy, immunotherapy, and conventional chemotherapy in CCA had certain mechanistic links, and the combination of those can greatly improve the prognosis of advanced CCA patients. This study aimed to review the research progress of targeted therapy and immunotherapy for CCA.

Targeting Epidermal Growth Factor Receptor to Stimulate Elastic Matrix Regenerative Repair.

Abdominal aortic aneurysms (AAAs) represent a multifactorial, proteolytic disorder involving disintegration of the matrix structure within the AAA wall. Intrinsic deficiency of adult vascular cells to regenerate and repair the wall elastic matrix, which contributes to vessel stretch and recoil, is a major clinical challenge to therapeutic reversal of AAA growth. In this study, we investigate the involvement of epidermal growth factor receptor-mitogen activated protein kinase (EGFR-MAPK) pathway in the activation of aneurysmal smooth muscle cells (SMCs) by neutrophil elastase, and how EGFR can be targeted for elastic matrix regeneration. We have demonstrated that neutrophil elastase activates EGFR and downregulates expression level of key elastin homeostasis genes (elastin, crosslinking enzyme-lysyl oxidase, and fibulin4) between a dose range of 1-10 μg/mL (p < 0.05). It also incites downstream proteolytic outcomes by upregulating p-extracellular signal-regulated kinase (ERK)1/2 (p < 0.0001) and matrix metalloprotease 2 (MMP2) at a protein level, which is significantly downregulated upon EGFR-specific inhibition by tyrosine kinase inhibitor AG1478 (p-ERK1/2 and MMP2 [p < 0.05]). Moreover, we have shown that EGFR inhibition suppresses collagen amounts in aneurysmal SMCs (p < 0.05) and promotes robust formation of elastic fibers by enhancing its deposition in the extracellular space. Hence, the EGFR-MAPK pathway in aneurysmal cells can be targeted to provide therapeutic effects toward stimulating vascular matrix regeneration. Impact statement Proteolytic disorders such as aortal expansions, called abdominal aortic aneurysms (AAAs), are characterized by naturally irreversible enzymatic breakdown and loss of elastic fibers, a problem that has not yet been surmounted by existing tissue engineering approaches. In this work, we show, for the first time, how epidermal growth factor receptor (EGFR) inhibition provides downstream benefits in elastic fiber assembly and deposition in aneurysmal smooth muscle cell cultures. This work can open future possibilities for development of EGFR-targeted drug-based therapies not only for vessel wall repair in AAAs but also other proteolytically compromised elastic tissues.

Novel considerations on EGFR-based therapy as a contributor to cancer cell death in NSCLC.

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) represented by gefitinib and erlotinib are widely used in treating non-small cell lung cancer (NSCLC). However, acquired resistance to EGFR-TKI treatment remains a clinical challenge. In recent years, emerging research investigated in EGFR-TKI-based combination therapy regimens, and remarkable achievements have been reported. This article focuses on EGFR-TKI-based regimens, reviews the standard and novel application of EGFR targets, and summarizes the mechanisms of EGFR-TKI combinations including chemotherapy, anti-vascular endothelial growth factor monoclonal antibodies, and immunotherapy in the treatment of NSCLC. Additionally, we summarize clinical trials of EGFR-TKI-based combination therapy expanding indications to EGFR mutation-negative lung malignancies. Moreover, novel strategies are under research to explore new drugs with good biocompatibility. Nanoparticles encapsulating non-coding RNA and chemotherapy of new dosage forms drawn great attention and showed promising prospects in effective delivery and stable release. Overall, as the development of resistance to EGFR-TKIs treatment is inevitable in most of the cases, further research is needed to clarify the underlying mechanism of the resistance, and to evaluate and establish EGFR-TKI combination therapies to diversify the treatment landscape for NSCLC.

Preclinical Evidence that Arctigenin Effectively and Selectively Targets Clear Cell Renal Cell Carcinoma Via Suppressing EGFR and RhoA

Clear cell renal cell carcinoma (ccRCC) has poor clinical outcomes and necessitates new treatment options. Epidermal growth factor receptor (EGFR) is a potential therapeutic target, due to the associations with various carcinomas’ progression. Arctigenin, a natural compound of Arctium lappa, has been shown to display anticancer abilities in various carcinomas. Cellular assays and combination studies were conducted using arctigenin and anti-ccRCC drugs. In vivo efficacy of arctigenin was determined using ccRCC xenograft mouse model. Immunoblotting and biochemistry analysis were applied to investigate the signaling affected by arctigenin. Arctigenin inhibits growth, migration, and survival of ccRCC cells while sparing normal kidney cells. Arctigenin acts synergistically with 5-FU and sorafenib but not temsirolimus in inhibiting ccRCC cells. Synergism of arctigenin with 5-FU and sorafenib was further shown in ccRCC xenograft mouse model. The combination of arctigenin with clinical anti-RCC drugs completely inhibits tumor growth without tumor progression even for an extended time period. Mechanistically, arctigenin inhibits migration in a RhoA-dependent manner while inhibits growth via suppressing EGFR-mediated signaling pathways. Our findings suggest that arctigenin performs well to add to current treatment in ccRCC and confirm the value to target EGFR to improve therapy in RCC.

Towards Radiolabeled EGFR-Specific Peptides: Alternatives to GE11.

The human epidermal growth factor receptor (EGFR) is closely related to several cancer-promoting processes and overexpressed on a variety of tumor types, rendering it an important target structure for the imaging and therapy of several malignancies. To date, approaches to develop peptidic radioligands able to specifically address and visualize EGFR-positive tumors have been of limited success. Most of the attempts were based on the lead GE11, as this peptide was previously described to be a highly potent EGFR-specific agent. However, since it has recently been shown that GE11 exhibits an insufficient affinity to the EGFR in monomeric form to be suitable as a basis for the development of tracers based on it, in the present work we investigated which other peptides might be suitable as lead structures for the development of EGFR-specific peptidic radiotracers. For this purpose, we developed 68Ga-labeled radioligands based on the peptides D4, P1, P2, CPP, QRH, EGBP and Pep11, having been described before as EGFR-specific. In addition, we also tested three truncated versions of the endogenous EGFR ligand hEGF (human epidermal growth factor) with respect to their ability to specifically target the EGFR with high affinity. Therefore, chelator-modified labeling precursors of the mentioned peptides were synthesized, radiolabeled with 68Ga and the obtained radioligands were evaluated for their hydrophilicity/lipophilicity, stability against degradation by human serum peptidases, in vitro tumor cell uptake, and receptor affinity in competitive displacement experiments on EGFR-positive A431 cells. Although all NODA-GA-modified (NODA-GA: (1,4,7-triazacyclononane-4,7-diyl)diacetic acid-1-glutaric acid) labeling precursors could be obtained more or less efficient in yields between 5 and 74%, the 68Ga-radiolabeling proved to be unsuccessful for two of the three truncated versions of hEGF ([68Ga]Ga-8 and [68Ga]Ga-9), producing several side-products. For the other agents [68Ga]Ga-1-[68Ga]Ga-7, [68Ga]Ga-10 and [68Ga]Ga-11, high radiochemical yields and purities of ≥98% and molar activities of up to 114 GBq/µmol were obtained. In the assay investigating the radiopeptide susceptibilities against serum peptidase degradation, the EGBP-based agent demonstrated a limited stability with a half-life of only 66.4 ± 3.0 min, whereas the other tracers showed considerably higher stabilities of up to an 8000 min half-life. Finally, all radiotracer candidates were evaluated in terms of tumor cell internalization and receptor binding potential on EGFR-positive A431 cell. In these experiments, all developed agents failed to show an EGFR-specific tumor cell uptake or a relevant EGFR-affinity. By contrast, the positive controls tested under identical conditions, [125I]I-hEGF and hEGF demonstrated the expected high EGFR-specific tumor cell uptake (33.6% after 1 h, being reduced to 1.9% under blocking conditions) and affinity (IC50 value of 15.2 ± 3.3 nM). Thus, these results indicate that none of the previously described peptidic agents developed for EGFR targeting appears to be a reasonable choice as a lead structure for the development of radiopeptides for targeting of EGFR-positive tumors. Likewise, the tested truncated variants of the endogenous hEGF do not seem to be promising alternatives for this purpose.

Dual Targeting of EGFR and MTOR Pathways Inhibits Glioblastoma Growth by Modulating the Tumor Microenvironment.

Glioblastoma's (GBM) aggressive growth is driven by redundant activation of a myriad of signaling pathways and genomic alterations in tyrosine kinase receptors, such as epidermal growth factor receptor (EGFR), which is altered in over 50% of cases. Single agents targeting EGFR have not proven effective against GBM. In this study, we aimed to identify an effective anti-tumor regimen using pharmacogenomic testing of patient-derived GBM samples, in culture and in vivo. High-throughput pharmacological screens of ten EGFR-driven GBM samples identified the combination of erlotinib (EGFRi) and MLN0128 (a mammalian target of rapamycin inhibitor, or MTORi) as the most effective at inhibiting tumor cell viability. The anti-tumor activity of erlonitib+MLN0128 was synergistic and produced inhibition of the p-EGFR, mitogen-activated protein kinase (MAPK), and Phosphoinositide 3-kinase (PI3K) pathways in culture. Using an orthotopic murine model of GBM, we show that erlotinib+MLN0128 inhibited tumor growth in vivo and significantly prolonged the survival of tumor-bearing mice. Expression profiling of tumor tissues from treated mice revealed a unique gene signature induced by erlotinib+MLN0128, consisting of downregulation of immunosuppressive chemokines in the tumor microenvironment, including C-C motif chemokine ligand 2 (CCL2) and periostin. Lower periostin levels resulted in the inhibition of Iba1+ (tumor-promoting) macrophage infiltration of GBM xenografts. Taken together, our results demonstrate that pharmacological co-targeting of EGFR and MTOR using clinically available drugs represents an effective treatment paradigm for EGFR-driven GBMs, acting both by inhibiting tumor cell growth and modulating the immune tumor microenvironment.

Binding sites in the epidermal growth factor receptor are responsible for bisphenol S effects on trophoblast cell invasion

Bisphenol S (BPS) is an endocrine disrupting chemical and the second most abundant bisphenol detected in humans. We have recently demonstrated that in utero exposure to BPS reduces human placenta cell fusion by interfering with epidermal growth factor (EGF)-dependent EGF receptor (EGFR) activation. Our previous work suggests that this occurs via binding of BPS to the extracellular domain of EGFR. However, whether BPS directly binds to EGFR has not been confirmed. We evaluated the binding ability of BPA, BPF and BPS to EGFR to determine whether EGFR binding is a unique attribute of BPS. To test these hypotheses, we first exposed HTR-8/SVneo cells to BPS, BPA, or BPF, with or without EGF. When co-exposed to EGF, BPS, but not BPA nor BPF, reduced EGFR phosphorylation by ∼60%, demonstrating that only BPS can interfere with EGF-dependent EGFR activation. As this indicates that BPS binding to the extracellular domain is responsible for its effect, we performed a computational search for putative binding sites on the EGFR extracellular domain, and performed ligand docking of BPS, BPA, and BPF at these sites. We identified three sites where polar interactions between positively charged residues and the sulfonyl group of BPS could lead binding selectivity over BPA and BPF. To test whether EGFR mutations at the predicted BPS binding sites (Arg255, Lys454, and Arg297) could prevent BPS's interference on EGFR activation, mutations for each EGFR target amino acids (R255A, R297A, and K454A) were introduced. For variants with R297A or K454A mutations, BPS did not affect EGF-mediated EGFR phosphorylation or EGFR-mediated cell invasion, suggesting that these residues are needed for the BPS antagonism effect on EGFR. In conclusion, BPS, but not BPA or BPF, interferes with EGFR-mediated trophoblast cell functions through binding at Arg297 and Lys454 amino acid residues in the extracellular domain of EGFR.

Treating Head and Neck Cancer in the Age of Immunotherapy: A 2023 Update

Most patients diagnosed with head and neck squamous cell carcinoma (HNSCC) will present with locally advanced disease, requiring multimodality therapy. While this approach has a curative intent, a significant subset of these patients will develop locoregional failure and/or distant metastases. The prognosis of these patients is poor, and therapeutic options other than palliative chemotherapy are urgently needed. Epidermal growth factor receptor (EGFR) overexpression is an important factor in the pathogenesis of HNSCC, and a decade ago, the EGFR targeting monoclonal antibody cetuximab was approved for the treatment of late-stage HNSCC in different settings. In 2016, the anti-programmed death-1 (PD-1) immune checkpoint inhibitors nivolumab and pembrolizumab were both approved for the treatment of patients with recurrent or metastatic HNSCC with disease progression on or after platinum-containing chemotherapy, and in 2019, pembrolizumab was approved for first-line treatment (either as monotherapy in PD-L1 expressing tumors, or in combination with chemotherapy). Currently, trials are ongoing to include immune checkpoint inhibition in the (neo)adjuvant treatment of HNSCC as well as in novel combinations with other drugs in the recurrent/metastatic setting to improve response rates and survival and help overcome resistance mechanisms to immune checkpoint blockade. This article provides a comprehensive review of the management of head and neck cancers in the current era of immunotherapy.

Synthesis, Radiolabeling, and In Vitro and In Vivo Characterization of Heterobivalent Peptidic Agents for Bispecific EGFR and Integrin αvβ3 Targeting.

Radiolabeled heterobivalent peptidic ligands (HBPLs) are a highly promising compound class for the sensitive and specific visualization of tumors as they often exhibit superior properties compared to their monospecific counterparts and are able to concomitantly or complementarily address different receptor types. The combination of two receptor-specific agents targeting the epidermal growth factor receptor (EGFR) and the integrin αvβ3 in one HBPL would constitute a synergistic combination of binding motifs as these two receptor types are concurrently overexpressed on several human tumor types and are closely associated with disease progression and metastasis. Here, we designed and synthesized two heterobivalent radioligands consisting of the EGFR-specific peptide GE11 and αvβ3-specific cyclic RGD peptides, bearing a (1,4,7-triazacyclononane-4,7-diyl)diacetic acid-1-glutaric acid chelator for efficient radiolabeling and linkers of different lengths between both peptides. Both HBPLs were radiolabeled with 68Ga3+ in high radiochemical yields, purities of 96-99%, and molar activities of 36-88 GBq/μmol. [68Ga]Ga-1 and [68Ga]Ga-2 were evaluated for their log D(7.4) and stability toward degradation by human serum peptidases, showing a high hydrophilicity for both agents of -3.07 ± 0.01 and -3.44 ± 0.08 as well as a high stability toward peptidase degradation in human serum with half-lives of 272 and 237 min, respectively. Further on, the in vitro receptor binding profiles of both HBPLs to the target EGF and integrin αvβ3 receptors were assessed on EGFR-positive A431 and αvβ3-positive U87MG cells. Finally, we investigated the in vivo pharmacokinetics of HBPL [68Ga]Ga-1 by positron emission tomography/computed tomography imaging in A431 tumor-bearing xenograft mice to assess its potential for the receptor-specific visualization of EGFR- and/or αvβ3-expressing tumors. In these experiments, [68Ga]Ga-1 demonstrated a tumor uptake of 2.79 ± 1.66% ID/g, being higher than in all other organs and tissues apart from kidneys and blood at 2 h p.i. Receptor blocking studies revealed the observed tumor uptake to be solely mediated by integrin αvβ3, whereas no contribution of the GE11 peptide sequence to tumor uptake via the EGFR could be determined. Thus, the approach to develop radiolabeled EGFR- and integrin αvβ3-bispecific HBPLs is in general feasible although another peptide lead structure than GE11 should be used as the basis for the EGFR-specific part of the agents.