A bispecific antibody targeting EGFR and AXL delays resistance to osimertinib

Audit of Molecular Mechanisms of Primary and Secondary Resistance to Various Generations of Tyrosine Kinase Inhibitors in Known Epidermal Growth Factor Receptor-Mutant Non-small Cell Lung Cancer Patients in a Tertiary Centre

Background: Somatic mutations in the epidermal growth factor receptor (EGFR) are detected in nearly 15% of patients with lung adenocarcinoma. These mutations are critical for tumor development and maintenance; some but not all are sensitive to EGFR tyrosine kinase inhibitors (TKI). EGFR mutations fall into 3 major categories: 1) Exon 19 deletions and L858R which are detected in 85% of patients; 2) Atypical EGFR mutations including G719X, L861Q which are detected in 5 to 8% of patients and 3) Exon 20 insertion mutations which are also detected in 5 to 8% of patients. These different subtypes exhibit varying responses to TKI treatment, with osimertinib being the standard of care for patients with exon 19 deletion or L858R mutations. However, no currently-approved TKI is available for patients with Exon 20/EGFR mutant lung cancer. Method: Patient advocacy groups representing oncogene-driver mutations in lung cancer (ALK, EGFR, Exon 20, RET, ROS1) have assembled with the objectives of providing patient education/support and driving research into lung cancer’s causes and treatments. The EGFR Resisters represent over 775 patients from 26 countries. In an effort to understand mechanisms of resistance and develop new treatments, EGFR Resisters embarked on a collaboration with Dr. Pasi Jänne, the Addario Lung Cancer Medical Institute, the Bonnie J. Addario Lung Cancer Foundation, and Champions Oncology to develop patient-derived xenograft (PDX) models for EGFR mutant lung cancer. Result: In mid-2018 the EFGR Resisters assembled a coalition of key leaders in the lung cancer space to advance development of research models for EGFR mutant lung cancer. The IRB-approved study, “A Prospective Biospecimen Collection Study from Patients with EGFR mutant Tumors” was launched in November 2018 with a goal of developing at least ten EGFR mutant PDX models with full characterization including whole exome sequencing (WES) and RNA sequencing. Model development will be divided among three cohorts: 1) EGFR T790M patients who have progressed on osimertinib or other third-generation TKIs; 2) EGFR exon 19 del or L858R patients who have progressed on first-line osimertinib; 3) patients with Exon 20 insertion mutations (includes EGFR Exon 20 and HER2 Exon 20). The models will be made available to the scientific community. Conclusion: Understanding mechanisms of resistance in EGFR mutant lung cancer is high priority as most patients will ultimately develop resistance to currently-approved TKIs. Additionally, there is an urgent need to develop next-generation TKIs since some EGFR mutation subtypes do not respond to current therapies. By leveraging the skills and expertise of diverse partners, the EGFR Resisters have advanced an initiative to create a unique cohort of PDX models that will provide a rich resource for clinical and translational research to understand mechanisms of resistance and develop new therapies for patients with EGFR mutant lung cancer. Citation Format: Ivy B. Elkins, Jill Feldman, Anita Figueras, Teri Kennedy, Allen Lee, Ildiko Medve, Colleen Sturdivant, Pasi Janne, Tony Addario, Alicia Sable-Hunt, Bonnie Addario, David LeDuc, Amy Moore, Jennifer Jaskowiak, Maria Mancini. A prospective biospecimen collection study from patients with EGFR mutant tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4744.

Abbreviation: ALK = anaplastic lymphoma kinase, EGFR = epidermal growth factor receptor, NSCLC = non-small cell lung cancer, TKI = tyrosine kinase inhibitor

The non-small cell lung cancer (NSCLC) accounts approximately 85% of lung cancers and includes predominantly adenocarcinomas, which is the most common type and squamous cell carcinomas. The treatment options include surgery, radiation therapy, and chemotherapy and the decision depends on the patient’s medical status and stage of disease. From 1970 the standard first line treatment for most patients with unresectable NSCLC and good performance status was the use of a combination of chemotherapy regimens and usually cisplatin-based. The most common combination regimens in use at present are platinum based regimens with gemcitabine, with paclitaxel or docetaxel and with vinorelbine combinations. The addition of the recombinant humanized monoclonal antibody bevacizumab that binds to vascular endothelial growth factor (VEGF) to carboplatin and paclitaxel for the treatment of non-squamous advanced NSCLC has demonstrated to increase response rate (RR), progression free survival (PFS) and overall survival (OS) when compared to chemotherapy alone. Despite recent advances with approval of more active chemotherapeutic and anti-angiogenesis agents for stage IV NSCLC, standard therapy can provide only modest clinical benefits with significant toxicities when used in unselected patients. In 2004, the identification of somatic mutations in the epidermal growth factor receptor (EGFR) gene provided the first glimpse of a possible target for a treatment which could maximize clinical outcome in those patients who could benefit from a personalized therapy. Identifying mutations in oncogenes associated with non-squamous NSCLC can help determine which patients are more likely to benefit from a targeted therapy. Such oncogenes include EGFR, KRAS, and ALK. The presence of an EGFR mutation confers a more favorable prognosis and strongly predicts for sensitivity to EGFR tyrosine kinase inhibitors (TKIs) such as erlotinib, gefitinib, and afatinib. The use of EGFR TKIs is based upon the detection of these mutations. The incidence of EGFR mutations in tumors with non-small-cell histology ranges from ~15% in Caucasians to ~50% in East Asians; 95% of such mutations have been found in adenocarcinomas. Patients bearing EGFR mutations have shown favorable clinical outcomes even with conventional chemotherapy suggesting that EGFR may be a predictive and a prognostic factor. Activation of the EGFR protein stimulates protein tyrosine kinase, which leads to activation of signaling pathways associated with cell growth and survival. Both EGFR overexpression and activating mutations in the tyrosine kinase domain of the EGFR gene lead to tumor growth and progression. Erlotinib, gefitinib and afatinib are examples of EGFR TKIs that can prevent activation of the signaling pathways and improve RRs in selected NSCLC patients. These mutations which are associated with increased sensitivity to EGFR TKIs, predominate in never-smokers, females, and tumors with adenocarcinoma histology. The most common mutations associated with sensitivity to EGFR TKIs include exon 19 deletions and the L858R point mutation and they are associated with RRs of >70%. Other EGFR mutations like T790M and exon 20 insertion, have been associated with much lower response or acquired resistance to TKI’s. The predictive value of EGFR mutations for use of gefitinib has been strengthened by the results of three randomized phase III trials that specifically compared TKIs used as first-line therapy with traditional platinum-based chemotherapy in patients with advanced NSCLC. In 2009 the results of IRESSA Pan-Asia Study were presented. This trial included a big number of Asian ethnicity patients (1,217) who were never smokers or former light smokers with histologic diagnosis of adenocarcinoma. The trial demonstrated an improvement in PFS and RR, with no statistical difference in OS, with the use of gefitinib in EGFR-mutated tumors and better RR and PFS with standard chemotherapy in patients without mutations. The first phase III trial of gefitinib versus chemotherapy as initial treatment of recurrent or advanced NSCLC, based on selection of patients with known activating EGFR mutations was the WJTOG3405 trial, reported in 2010. This trial documented important achievements in RR and PFS with the use of TKIs. Almost the same results were confirmed by another similar Japanese phase III trial, NEJ002, with RR and PFS definitely favoring the use of gefitinib in the first-line setting of metastatic EGFR-mutated NSCLC. Based on the results of the IPASS study, gefitinib was approved for use in Europe for the initial treatment of patients with NSCLC exhibiting EGFR mutations. The positive results of the EURTAC trial, NCT00446225, which was a randomized phase III trial of erlotinib versus standard chemotherapy, suggested that responsiveness in mutation-positive patients was not a function of ethnicity. Afatinib is approved as monotherapy for the treatment of EGFR TKI—naïve adults with locally advanced or metastatic NSCLC with activating EGFR mutations in the EU, and for the first-line treatment of patients with metastatic NSCLC whose tumors have EGFR exon 19 deletions or exon 21 (L858R) substitution mutations as detected by a US FDA-approved test in the US. In two randomized, open-label, multinational phase III trials, progression-free survival was significantly prolonged with afatinib compared with pemetrexed plus cisplatin (LUX-Lung 3) or gemcitabine plus cisplatin (LUX-Lung 6) in treatment-naïve patients with advanced NSCLC with activating EGFR mutations. EGFR-TKIs as a class are generally well tolerated. The two most common toxicities include dermatologic and GI effects, which are mild to moderate, easily managed and reversible. In order to determine whether an EGFR TKI or chemotherapy is the appropriate first-line therapy, the latest guidelines recommend mutation testing for all patients with advanced NSCLC tumor. The aim of this prospective study is to compare the efficacy of gefitinib, erlotinib and afatinib in patients with advanced NSCLC harboring activating EGFR mutations in first line of treatment. These agents are recommended as first line treatments for NSCLCs with such mutations. The primary endpoint will be the PFS and the secondaries will be the OS and the record of the toxicities. In each of the 3 arms will be participate 20 patients with EGFR mutated tumors. The technique for screening NSCLC patients for driver mutations that it will be used is next-generation sequencing, which overcomes many of the shortcomings of direct sequencing. This massively parallel approach, relying heavily on automation, data storage, and computational processing, allows quantitative analysis of infrequent alleles and simultaneous evaluation of multiple genes or even whole genomes, but is not yet used routinely in clinical practice. In addition, KRAS mutation analysis will be performed in patients with known smoking history in order to determine the correlation of type and mutation frequency with smoking.

Non-small cell lung cancers (NSCLCs) with activating mutations in the Epidermal Growth Factor Receptor (EGFR) gene are associated with high response rates (70-80%) to EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib and gefitinib, but most acquire resistance over time through numerous mechanisms. In these patients, the cMet pathway is often activated to compensate and provide resistance to the EGFR targeted monotherapy; this activation can occur by MET gene amplification, overexpression of cMet protein, or an increase in the ligand HGF. We have designed a bispecific EGFR-cMet antibody (JNJ-61186372) with multiple mechanisms of action resulting in anti-tumor activity in the EGFR mutant setting, with or without cMet pathway activation. Controlled Fab-arm exchange was used to produce JNJ-61186372, a technique that allows for efficient large-scale preparation of bispecific antibodies with a regular IgG1 structure. JNJ-61186372 was shown to bind EGFR and cMet and efficiently inhibited ligand-induced phosphorylation of both receptors. In addition to this important mechanism of action, we have engineered the antibody to contain lower than normal fucose levels to increase Fc-dependent effector mechanisms. JNJ-61186372 exhibited antibody dependent cellular cytotoxicity (ADCC) activity in vitro in a range of NSCLC cell lines with EGFR mutations, KRas mutation, and/or amplified MET gene. Furthermore, the low fucose form of JNJ-61186372 demonstrated more effective ADCC activity compared to its normal fucose counterpart. The bispecific JNJ-61186372 antibody showed increased potency (2-3 fold) compared to the combination of monovalent EGFR and monovalent cMet antibodies, demonstrating the beneficial effects of dual targeting in a single molecule. Antibody dependent cell-mediated phagocytosis (ADCP) activity of JNJ-61186372 was also confirmed in vitro. We have also demonstrated that Fc-dependent effector functions contributed to in vivo anti-tumor growth activity of JNJ-61186372, in a xenograft model with EGFR mutations and cMet activation. Our data demonstrate that the bispecific antibody JNJ-61186372, generated using controlled Fab-arm exchange, has in vitro ADCC and ADCP activity in EGFR mutant settings, either with or without cMet pathway activation, and with KRas mutation. In addition, the Fc-dependent effector mechanisms contributed to in vivo anti-tumor efficacy. The dual signaling inhibition of EGFR and cMet pathways by JNJ-61186372, combined with enhanced Fc effector function, may provide multiple mechanisms to combat resistance in EGFR mutant NSCLC patients. Citation Format: Keri L. Soring, Katharine D. Grugan, Randall J. Breszki, Jose Pardinas, Leopoldo Luistro, Barbara Bushey, Joost Neijssen, Paul Parren, Janine Schuurman, Mark Anderson, Ricardo Attar, Matthew V. Lorenzi, Mark Chiu, Sheri Moores. Activity of a bispecific antibody targeting EGFR and cMet with enhanced Fc effector function in EGFR mutant setting with cMet pathway activation. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A11.

Commentary: Another win for immunotherapy

The murine retroviral oncogene v-cbl induces pre-B cell lymphomas and myelogenous leukemias. The protein product of the mammalian c-cbl proto-oncogene is a widely expressed cytoplasmic 120-kDa protein (p120cbl) whose normal cellular function has not been determined. Here we show that upon stimulation of human epidermal growth factor (EGF) receptor, p12ocbl becomes strongly tyrosine-phosphorylated and associates with activated EGF receptor in vivo. A GST fusion protein containing amino acids 1-486 of p120cbl, including a region highly conserved in nematodes, binds directly to the autophosphorylated carboxyl-terminal tail of the EGF receptor. Platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), or nerve growth factor (NGF) stimulation also results in tyrosine phosphorylation of p120cbl. Recent genetic studies in Caenorhabditis elegans indicate that Sli-1, a p120cbl homologue, plays a negative regulatory role in control of the Ras signaling pathway initiated by the C. elegans EGF receptor homologue. Our results indicate that p120cbl is involved in an early step in the EGF signaling pathway that is conserved from nematodes to mammals.

Background: Epidermal growth factor receptor (EGFR) inhibition with tyrosine kinase inhibitors (TKIs) has proven successful in the management of patients with non small cell lung cancer (NSCLC). However, clinical benefit is predominantly limited to the subgroup of patients with an activating EGFR mutation (10%). Treating the remaining 90% of patients with wild type (wt) EGFR is an unmet clinical need. Aim: To evaluate the effect of an allosteric AKT inhibitor (AKTi 1/2) in combination with the EGFR inhibitor gefitinib on cell proliferation, apoptosis and signalling output in EGFR mutant/wt NSCLC cell lines. Methods: Four NSCLC cell lines were selected: NCI-H522 and NCI-H1651 (EGFR wt and K-RAS wt), PC9 and HCC827 (EGFR mutant). The 96hr sulphorhodamine assays was used to assess the effect of gefitinib and AKTi 1/2 on growth inhibition, and median effect analysis to calculate combination indices (CIs) to assess the effect of both drugs in combination. The expression of p-EGFR, p-AKT, p-S6, p-ERK and cleaved PARP were studied by Western blotting in the EGFR wt NCI-H522 and the EGFR mutant PC9 cell lines. Results: EGFR wt cell lines NCI-H522 and NCI-H1651 were relatively resistant to gefitinib (IC50 values of 7.0 ± 2.5 uM and 8.8 ± 0.9 uM, respectively) compared with the EGFR mutant cell lines PC9 and HCC827 (IC50 values of 0.07 ± 0.03 uM and 0.004 ± 0.0005 uM, respectively). The combination of gefitinib and AKTi 1/2 produced synergistic inhibition of growth in both EGFR wild type and mutant cell lines. Synergism was more marked in EGFR wt cell lines with CI values (ED50) of 0.53 ± 0.28 and 0.49 ± 0.17 for NCI-H522 and NCI-H1651 respectively, compared with 0.74 ± 0.11 and 0.76 ± 0.14 for the EGFR mutant cell lines PC9 and HCC827, respectively. Concomitant exposure of cells to gefitinib and AKTi 1/2 for 24 hrs caused incremental inhibition of p-AKT, p-S6 and p-ERK in both the EGFR wt (NCI-H522) and EGFR mutant (PC9) cell lines. Gefitinib alone resulted in partial inhibition of AKT phosphorylation in PC9 cells and up-regulation in NCI-H522 cells. Incremental induction of cleaved PARP, a marker of apoptosis, was seen in PC9 cells treated with the combination of gefitinib and AKTi 1/2. Conclusions: These results demonstrate synergistic growth inhibition of EGFR wt and mutant NSCLC cell lines by the combination of gefitinib with AKTi 1/2. Additional pre-clinical studies are investigating this further. Clinical studies of the combination doublet are warranted to expand the utility of EGFR TKI in EGFR wt NSCLC. Citation Format: Martina Puglisi, Parames Thavasu, Adam Stewart, Jaishree Bhosle, Sanjay Popat, Mary E R O'Brien, Udai Banerji. Evaluation of combination of an EGFR and AKT inhibitor in EGFR mutant and EGFR wild type non-small cell lung cancer cells lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2441. doi:10.1158/1538-7445.AM2013-2441

Six first-line tyrosine kinase inhibitors reveal novel inhibition potential for the EGFR S768I mutation

Chapter 33 - EGFR Mutations: Best Results from Second- and Third-Generation Tyrosine Kinase Inhibitors

Meta-Analysis of EGFR Kinase Inhibitors: Not Always Greater Than the Sum of Its Parts

Evolving treatment for advanced non-small cell lung cancer harbouring common EGFR activating mutations.

BackgroundNon-surgical cytological specimens are adequate not only for accurate histological subtyping but also for molecular profiling. A modified amplification refractory mutation system polymerase chain reaction (ARMS PCR), known as SuperARMS PCR, was improved by optimizing the primers designation, which provides a higher sensitivity and specificity approach for free plasma DNA detection. It is unclear whether SuperARMS PCR detects epidermal growth factor receptor (EGFR) mutations in cytology samples. The aim of this study was to compare the EGFR mutations detected by ARMS PCR and SuperARMS PCR in cytology samples derived from advanced non-small cell lung cancer (NSCLC) patients.MethodsFrom March 2016 to March 2018, a total of 234 cytological samples were obtained from primary or metastatic lesions of NSCLC, including 144 fine-needle aspirations (FNAs), 36 endobroncheal ultrasonography (EBUS) FNAs, 36 transbronchial needle aspirations (TBNAs) and 18 pleural effusion (PLEs). EGFR mutations were simultaneously detected using an ADx-ARMS EGFR kit (Amoy Diagnostics CO., ltd., Xiamen, China) and an ADx-SuperARMS EGFR kit (Amoy Diagnostics CO., ltd., Xiamen, China). Digital droplet PCR (ddPCR) and next-generation sequencing (NGS) were further used to verify the EGFR mutant inconsistent samples.ResultsAll of the 234 patients with advanced or recurrent NSCLC were diagnosed and assessed by two cytopathologists, and their EGFR mutation statuses were successfully detected by ARMS and SuperARMS. Importantly, the SuperARMS and ARMS methods showed a highly concordant result of 94.0% (220/234) (95%CI: 85.0, 95.0%). The positive rate of the SuperARMS was higher than the ARMS in the cytology samples for EGFR detection (46.2% vs. 40.2%). The specific EGFR mutation sites in 16 samples (6.8%) were not completely consistent between the SuperARMS and ARMS. A total of 14 patients showed EGFR mutations when detected by SuperARMS, but by ARMS there were EGFR wild-type. Two patients were detected as having one more EGFR mutation site by SuperARMS than by ARMS. ddPCR and NGS were used to further confirm the EGFR mutations in these inconsistent samples. Eight samples had the same mutation results as the SuperARMS, and 6 samples were not verified because the remaining DNA was insufficient. A total of 78 EGFR mutation patients received Tyrosine Kinase Inhibitor (TKI) treatment. The overall objective response rate (ORR) was 88.5% (69/78) for EGFR TKI treatment.ConclusionSuperARMS showed a high sensitivity and specificity for EGFR detection and thus, is expected to become a routine test in the clinic to be used as a widely available, easy-to-operate and sensitive method for EGFR mutation detection in liquid-based cytology samples.

The epidermal growth factor receptor (EGFR) is a cell-surface receptor tyrosine kinase that belongs to the ERBB family, which includes HER1 (EGFR), HER2, HER3, and HER4. Each of these receptors has three key layers: an input layer, a signal-processing core, and an output layer. EGFR is activated primarily through ligand binding, with ligands such as EGF, TGF-alpha, HB-EGF, AR, EPR, BTC, and epigen. However, mutations or overexpression of EGFR can lead to ligand-independent activation. Once activated, EGFR triggers various downstream signaling pathways, such as PI3K-AKT, JAK-STAT, RAS-MEK/ERK, and PLC-gamma-PKC, which enhance cell proliferation, survival, and resistance to cell death.The overexpression or increased activity of EGFR is linked to several cancers, including non-small cell lung cancer (NSCLC), colon cancer, head and neck cancer (HNC), pancreatic cancer, breast cancer, and brain cancer. Recognizing EGFR as a driver gene has accelerated the development of targeted anticancer therapies, such as monoclonal anti-EGFR antibodies (cetuximab, panitumumab) and small molecule receptor tyrosine kinase inhibitors (TKIs). The first generation of EGFR TKIs, like gefitinib and erlotinib, specifically target mutations such as L858R and exon 19 deletions. To address resistance to these early inhibitors, second-generation EGFR TKIs (afatinib, dacomitinib) were developed. The EGFR T790M mutation is a major mechanism of resistance to both first and second-generation TKIs. Osimertinib, a third-generation TKI, was approved for use in EGFR-mutated NSCLC following the failure of first- and second-generation TKIs, although the EGFR C797S mutation limits its effectiveness. Fourth-generation EGFR TKIs, including BLU-945 and BBT-176, are under clinical evaluation but are not yet approved.We have created more than 200 Ba/F3-EGFR engineered cell lines, making this the largest in vitro and in vivo platform for drug discovery with the widest range of mutant cells. These cell lines include almost all clinically significant EGFR mutations, such as common mutations like L858R and exon 19 deletions, as well as rare mutations like L861Q, S768I, and combinations of mutations like L858R/T790M, Del19/T790M, and triple mutations. These Ba/F3-EGFR engineered cell lines are valuable tools for developing and assessing next-generation EGFR inhibitors and for studying newly acquired resistance mutations in EGFR. Citation Format: Yao Tang, Guoqian Wang, Tingduo Lv, Jingxiao Xu, Yao Peng, Jinying Ning, Feng Hao. Over 200! The largest BaF3-EGFR engineering cell lines collection, a useful platform for novel drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5981.

Deletion of the epidermal growth factor receptor in renal proximal tubule epithelial cells delays recovery from acute kidney injury