Targeting Epidermal Growth Factor Research Articles

Abstract 5423: Translational PK/PD/efficacy modeling and efficacious human dose prediction for a first-in-class MUC1-EGFR (M1231) bispecific antibody drug conjugate

Abstract Introduction: M1231 is a first-in-class bispecific antibody drug conjugate (ADC) targeting mucin-1 (MUC1) and epidermal growth factor receptor (EGFR), that is conjugated with a novel hemiasterlin-related microtubule inhibitor payload. Following dose, M1231 binds to EGFR and co-binds to tumor-associated hypoglycosylated MUC1, internalizes into the tumor cell and traffics to lysosomes where the payload is enzymatically released to affect cell viability. Methods: A Multi-scale Systems Pharmacology model was developed to account for the ADC’s disposition and interactions with the underlying physiologic system, resulting in intracellular payload release in tumor cells to drive tumor growth inhibition (TGI). An in vitro model quantified ADC internalization and lysosomal trafficking in the MUC1 and EGFR-expressing cancer cell lines MDA-MB-468 and OVCAR-3. TGI was assessed in mice bearing MUC1-tumors from the squamous NSCLC patient-derived xenograft model, LUX003. Pharmacokinetics in cynomolgus monkeys was modeled using a Target Mediated Drug Disposition model and was allometrically scaled to humans. Data Summary: All preclinical modeling results were integrated and scaled to simulate plasma M1231 concentrations to predict the human dose and corresponding exposures. Tumor stasis was estimated to begin at a dose of 2.4 mg/kg every 3 weeks (Q3W) with a maximum tumor regression achieved at a dose of 4.3 mg/kg Q3W. Conclusions: The quantitative systems pharmacology model-based efficacious dose prediction range of 2.4 mg/kg to 4.3 mg/kg dosed Q3W informed the design of the ongoing M1231 first-in-human trial (NCT04695847). Citation Format: Anup Zutshi, Berend Neuteboom, Seema Kumar, Willem Sloot, Christine Knuehl, Julia Dotterweich, Jianguo Ma, Christiane Amendt, Karthik Venkatakrishnan, Taeshin Park, John Pappas, Kyoung-Ae Kim. Translational PK/PD/efficacy modeling and efficacious human dose prediction for a first-in-class MUC1-EGFR (M1231) bispecific antibody drug conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5423.

Binding Potential of Dysidea Herbacea Derived Small Molecules to Breast Cancer Implicating Targets Estrogen and Epidermal Growth Factor Receptors

Identifying new targets and new drugs has always been a daunting task, especially in cancer research. This studyexamines the binding interaction and the drug-likeness properties of small molecules derived from marine sponge Dysideaherbacea to breast cancer receptors: epidermal growth factor receptor and estrogen receptor. The receptor’s interaction with the ligand was evaluated using the Schrodinger Glide package, and affinities were assessed based on the glide score. Ligand molecules that have higher binding affinity were evaluated further for their ADMET properties using Molinspiration.We found multiple ligands binding to these targets; however, Pyridine 3 carboxyamidewas found to have binding affinity to both the receptors. Compared to the other small molecules,further simulation studies could be taken up to ascertain its structural dynamics and ensuing in-vitro experiments that could prove growth inhibition of breast cancer cells.

Dual EGFR blockade with cetuximab and erlotinib combined with anti-VEGF antibody bevacizumab in advanced solid tumors: a phase 1 dose escalation triplet combination trial

BackgroundAngiogenesis and activation of the epidermal growth factor (EGFR) pathway play an essential role in tumor proliferation and metastasis. Targeting angiogenesis or EGFR alone does not yield adequate tumor control in most solid tumors. Overcoming intrinsic and/or acquired resistance may need a doublet or triplet therapy strategy. Herein, we report the safety and feasibility of dual EGFR blockade with EGFR monoclonal antibody and EGFR tyrosine kinase inhibitor combined with anti-VEGF antibody in advanced solid tumors.MethodsWe conducted a phase I study combining erlotinib, cetuximab, and bevacizumab. Patients with advanced or metastatic solid tumors (excluding colorectal and non-small cell lung cancers) were analyzed for safety, toxicity profile, and response. Anti-tumor activity was evaluated per response evaluation criteria in solid tumors (RECIST 1.0).ResultsThirty-six patients received treatment on a range of dose-levels. The most frequent tumor types enrolled were cervical (n = 10), head and neck squamous cell (n = 10), and follicular thyroid (n = 4) cancers. The most common treatment-related grade ≥ 2 adverse events were rash (56%), hypomagnesemia (17%), pruritus (11%), diarrhea (8%), and tumor-related bleeding (8%). Seventeen of 19 patients (89%) treated at the maximum tolerated dose did not present treatment-related dose-limiting toxicity. Fifteen (63%) of the 24 evaluable patients achieved a disease control (stable disease ≥ 4 months (n = 14) and partial response (n = 1). The median number of prior lines of therapies was 3 (range 1–10).ConclusionsThe triplet combination of erlotinib, cetuximab, and bevacizumab was well tolerated, conferring clinical benefit in heavily pretreated patients. Future studies are warranted with second or third-generation EGFR tyrosine kinase triplet combinations in the EGFR pathway aberrant patients.Trial Registration: ClinicalTrials.gov Identifier: NCT00543504. Sponsor(s): National Cancer Institute (NCI), MD Anderson Cancer Center

501P - Oncogenic mutations at the dimer interface of EGFR lead to formation of covalent homo-dimers and allosteric activation of the kinase domain: A mechanism which alters the selectivity profile of oncogenic EGFR

BackgroundMutation of either the intracellular catalytic domain or the extracellular domain of the receptor for epidermal growth factor (EGFR) drives oncogenicity. Extracellular domain EGFR mutations are highly expressed in patients with glioblastoma. Despite clinical success with targeting EGFR catalytic site mutants, no drugs have proven effective in glioblastoma patients expressing extracellular EGFR mutations. MethodsHerein, we define the molecular mechanism for oncogenic activation of families of extracellular EGFR mutations and reveal how this mechanism renders current generation small molecule ATP-site inhibitors ineffective. We define these types of lesions as allosteric oncogenic mutations. ResultsWe demonstrate that a group of the most commonly expressed extracellular domain EGFR mutants expressed in glioblastomas is activated by disulfide-bond mediated covalent homodimerization, collectively referred to as locked dimerization (LoDi-EGFR oncogenes). Strikingly, current generation small molecules binding to the active kinase conformation potently inhibit catalytic site mutants, but induce covalent dimerization and activate LoDi-EGFR receptors, manifesting in paradoxical acceleration of proliferation. Herein we describe the discovery of novel ATP competitive small molecules that potently (<50nM) inhibit the family of LoDi-EGFR oncogenes expressed in GBM, and importantly, achieve selectivity versus WT-EGFR of greater than 10-fold. ConclusionsThese data demonstrate how the locked-dimer mechanism of EGFR oncogenesis has profound impact on the activity of small molecules acting at the distal catalytic site, providing further evidence for “inside-out” allosteric signaling in EGFR. This provides a mechanistic understanding for the failure of current generation EGFR inhibitors to effectively treat LoDi-EGFR mutants in GBM and sets guidelines for the discovery and development of novel selective LoDi-EGFR inhibitors. Legal entity responsible for the studyBlack Diamond Therapeutics. FundingBlack Diamond Therapeutics. DisclosureE. Buck: Leadership role, Shareholder / Stockholder / Stock options, Full / Part-time employment: Black Diamond Therapeutics. M. O’Connor: Leadership role, Shareholder / Stockholder / Stock options, Full / Part-time employment: Black Diamond Therapeutics. A. Flohr: Leadership role, Shareholder / Stockholder / Stock options: Black Diamond Therapeutics. R. Iacone: Leadership role, Shareholder / Stockholder / Stock options: Black Diamond Therapeutics. A.V. Mayweg: Leadership role, Shareholder / Stockholder / Stock options, Officer / Board of Directors: Black Diamond Therapeutics. D.M. Epstein: Leadership role, Shareholder / Stockholder / Stock options, Officer / Board of Directors: Black Diamond Therapeutics. All other authors have declared no conflicts of interest.

Abstract LB-111: Epidermal growth factor receptor oncogenes expressed in glioblastoma are activated as covalent dimers and exhibit unique pharmacology

Abstract Mutation of either the intracellular catalytic domain or the extracellular domain of the receptor for epidermal growth factor (EGFR) drives oncogenicity. Extracellular domain EGFR mutations are highly expressed in patients with glioblastoma. Despite clinical success with targeting EGFR catalytic site mutants, no drugs have proven effective in glioblastoma patients expressing extracellular EGFR mutations. Herein, we define the molecular mechanism for oncogenic activation of families of extracellular EGFR mutations and reveal how this mechanism renders current generation small molecule ATP-site inhibitors ineffective. We demonstrate that a group of the most commonly expressed extracellular domain EGFR mutants expressed in glioblastomas is activated by disulfide-bond mediated covalent homodimerization, collectively referred to as locked dimerization (LoDi-EGFR oncogenes). Strikingly, current generation small molecules binding to the active kinase conformation potently inhibit catalytic site mutants, but induce covalent dimerization and activate LoDi-EGFR receptors, manifesting in paradoxical acceleration of proliferation. These data demonstrate how the locked-dimer mechanism of EGFR oncogenesis has profound impact on the activity of small molecules acting at the distal catalytic site, providing further evidence for “inside-out” allosteric signaling in EGFR. This provides a mechanistic understanding for the failure of current generation EGFR inhibitors to effectively treat LoDi-EGFR mutants in GBM and sets guidelines for discovery of selective LoDi-EGFR inhibitors. Citation Format: Matthew O'Connor, Theodore Nicolaides, Jie Zhang, Alexander Flohr, Roberto Iacone, Alexander V. Mayweg, David M. Epstein, Elizabeth Buck. Epidermal growth factor receptor oncogenes expressed in glioblastoma are activated as covalent dimers and exhibit unique pharmacology [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 LB-111.

PO-132 Alpha5 beta1 integrin provides glioma cell resistance to EGFR tyrosine kinase inhibitors. role of membrane trafficking

IntroductionOverexpression of epidermal growth factor (EGFR) drives glioblastoma (GBM) cell invasion and tumour progression. EGFR has been a major therapeutic target in GBM. However, clinical trials were disappointing, and we are still missing molecular basis to explain these poor results. In other solid tumours, extracellular matrix proteins found in the tumour microenvironment and their heterodimeric integrin receptors trigger resistance to EGFR targeted therapy. We recently established that the fibronectin receptor, α5β1 integrin is a pertinent therapeutic target in GBM. Therefore, we seek to evaluate whether α5β1 integrin is involved in therapy targeting EGFR in GBM.Material and methodsWe used GBM cell line (U87) overexpressing or down expressing α5 integrin subunit. EGFR was inhibited by clinically approved tyrosine kinase inhibitors (TKIs) (gefitinib, erlotinib, lapatinib). For cell motility assays, we performed spheroid dissemination assays, in 2D and 3D environment and chemotaxis assays through Boyden chambers. We also evaluated the impact of integrin expression on cell clonogenicity and cell grow th (2D, spheroids) in presence of EGFR inhibitors. Confocal microscopy confirmed integrin, EGFR and endosomes markers localization. Protein colocalization in early-endosomes was confirmed by confocal image analysis and dSTORM super-resolution.Results and discussionsWe showed that loss of integrin α5 expression increased U87 cell sensitivity to TKIs in spheroid cell evasion assays but did not affect TKIs efficiency on chemotaxis, cell growth (2D or 3D) or cell clonogenicity. EGFR is thus critical for cell dissemination from GBM tumour spheroïds and overexpression of α5 integrin can circumvent EGFR inhibition by TKIs. Using confocal imaging, we showed that TKIs provoked a massive relocalization of integrin and EGFR in Rab5, EEA1 or LRP1 positive endosomes. Dynamic studies confirmed that TKIs increased EGFR receptor endocytosis. By colocalization studies and super-resolution dSTORM imaging, we clearly showed that in intracellular vesicules integrin and EGFR are in close proximity, suggesting a potential interaction.ConclusionWe showed in GBM cells that α5β1 integrin expression triggers resistance to EGFR-targeting TKIs and that TKIs alter EGFR and integrin membrane trafficking. These data point out the potential importance of integrin and EGFR endocytosis and trafficking in GBM resistance to TKIs.

Abstract A203: CD3-EGFR bispecific Probody™ therapeutics induced tumor regressions and increased therapeutic window in preclinical studies

Abstract T cell-engaging bispecific antibodies (TCBs) represent a highly potent modality to direct the activity of cytotoxic T cells to tumors. TCBs are designed to bind both a surface tumor antigen and the CD3ϵ subunit of the T cell receptor, thereby enabling T cells to kill tumor cells. Because T cell bispecifics bypass TCR antigen recognition and co-stimulatory requirements for T cell activation, they obviate the need for tumor-specific immunity and overcome many impediments faced by T cells in the tumor-microenvironment. TCBs have shown clinical activity in hematologic malignancies as evidenced by the recent approval of blinatumomab for the treatment of relapsed refractory acute lymphoblastic leukemia based on its ability to direct T cells against CD19+ cells. Development of TCBs for non-hematologic cancers is proving more challenging, however, due to the high potency of the modality and its inability to discriminate between cells expressing target antigen in the tumor and those in healthy tissue. Because antigens with highly restricted tumor expression are rare, toxicity directed against healthy tissues has limited the therapeutic utility of TCBs for most solid tumor targets. In addition, because TCBs are administered at very low doses, preferential distribution to circulating T cells or to healthy tissues can limit drug exposure in tumors. Therefore new methods are needed that enable the potent anti-tumor activity of TCBs without on-target damage to normal tissues. Here we describe a T cell-engaging Bispecific Probody therapeutic (TCBP) targeting CD3 and Epidermal Growth Factor Receptor (EGFR). Probody therapeutics are recombinant, proteolytically-activated antibody prodrugs designed to widen therapeutic window by minimizing interaction with normal tissues and maximizing interactions with tumors. A Probody therapeutic is an antibody in which the antigen-binding site is blocked by a masking peptide extension from the amino terminus of the light chain. The peptide mask, joined to the antibody by a protease-cleavable linker, can be removed by proteases that are more active in the tumor microenvironment than in normal tissue, allowing for tumor-localized activity. We have previously demonstrated that an EGFR-targeted Probody therapeutic limits interaction with EGFR-expressing tissue while preserving anti-tumor activity. To demonstrate that Probody technology can address the limitations of bispecific antibodies, we compared CD3-EGFR TCBPs to the corresponding unmasked TCB for their ability to direct T cell-mediated cytotoxicity of EGFR+ tumor cells in vitro. While the unmasked CD3-EGFR TCB demonstrated potent T cell- and EGFR-dependent killing at single-digit pM concentrations, the CD3-EGFR TCBP exhibited reduced targeted cytotoxicity by up to 3000-fold. In a tumor model utilizing engraftment of human T cells in NSG mice, both TCB Probody therapeutic and its parental TCB effectively eliminated established EGFR+ HT-29 colorectal tumors at equivalent doses. In contrast, an ongoing study in non-human primates has shown at least a 10-fold higher maximum tolerated dose for the CD3-EGFR TCBP relative to its corresponding CD3-EGFR TCB. As a result, Probody technology has the potential to increase therapeutic window and may enable potent bispecific therapies for solid tumors that are limited by on-target toxicities in healthy tissue. Citation Format: Sherry L. LaPorte, Daniel R. Hostetter, Laurie Wong, Jennifer Razo, Linnea Diep, Clayton W. White, Jennifer H. Richardson, W. Michael Kavanaugh, Bryan A. Irving. CD3-EGFR bispecific Probody™ therapeutics induced tumor regressions and increased therapeutic window in preclinical studies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A203.

ASCO 2015: Highlights in breast cancer

Several important studies were presented at the 2015 ASCO Annual Meeting in the field of breast cancer. Austrian Breast and Colorectal Study Group trial 18 again indicated the importance of bone health in postmenopausal women receiving endocrine therapy and showed that denosumab, a monoclonal antibody targeting the RANK ligand, was highly active in the reduction of fracture rates in this population. In the metastatic setting, addition of palbociclib to fulvestrant nearly tripled progression-free survival in pretreated women with luminal breast cancer as shown in the randomized phase III PALOMA-3 trial. In HER2-positive breast cancer, results from a randomized phase II study suggested that T-DM1 with or without endocrine therapy may be a valuable option as neoadjuvant treatment. In this trial, pathologic complete remission rates of > 40 % were observed after only four cycles of T-DM1. The phase III ExteNET study evaluated the role of neratinib, a tyrosine-kinase inhibitor targeting HER2 and epidermal growth factor receptor, as extended adjuvant therapy after 1 year of trastuzumab; while the future role of neratinib remains still undefined, this was the first study to show an improvement in terms of disease-free survival over the current treatment standard. Finally, the MARIANNE trial showed that T-DM1 was as effective as trastuzumab plus docetaxel as first-line therapy, while dual HER2-inhibition with T-DM1 plus pertuzumab yielded no further benefit.

Targeting MET and EGFR in NSCLC-what can we learn from the recently reported phase III trial of onartuzumab in combination with erlotinib in advanced non-small cell lung cancer?

The epidermal growth factor (EGFR) is overexpressed in non-small cell lung cancer (NSCLC), and treatment with erlotinib as monotherapy in patients previously treated with chemotherapy is associated with improved overall survival (OS) (1). However, resistance to tyrosine kinase inhibitors targeting the EGFR is observed in the majority of patients and may be associated with the overexpression and/or amplification of the tyrosine kinase receptor MET (2). Increased MET expression in NSCLC has also been associated with worse prognosis and is co-expressed in 70% of EGFR mutant tumours (2). This data generated a valid rationale for combining MET and EGFR targeted agents in the management of advanced NSCLC.

Treatment with bevacizumab (BEV) upregulates expression of the transcription factor Sp1 and its downstream target genes in human carcinoid cells: Molecular basis of the synergistic antiangiogenic activity of bevacizumab and mithramycin A (MIT)

15031 Background: Our previous studies show that human carcinoid cells overexpress pro-angiogenic factors, vascular endothelial growth factor A (VEGF), and transcription factor Sp1 plays a critical role in VEGF inducible and constitutive expression. However, the impact of antiangiogenic therapy on the Sp1/VEGF pathway remains unclear. Method: Groups of 10 athymic BALB/c nude mice were implanted with 1.5 million human H727 carcinoid cells. Treatment with VEGF neutralizing monoclonal antibody, BEV, MIT, or BEV + MIT was initiated once implanted tumor reached 4 mm in size. Result: Treatment with BEV, suppressed human carcinoid growth in nude mice (tumor size at week 5 1280 mm3 vs 480 mm3; p &lt; 0.001). Gene expression analyses revealed that this treatment substantially upregulated the expression of Sp1 (7 folds) and its downstream target genes, including VEGF (5 folds) and epidermal growth factor receptor (4 folds), in tumor tissues, whereas it did not have this effect on carcinoid cells in culture. Treatment with mithramycin A, an Sp1 inhibitor, suppressed the expression of Sp1 and its downstream target genes in both cell culture and tumors growing in nude mice. Median survival of mice treated with PBS, BEV, MIT, and BEV + MIT groups were 88, 112, 121, and &gt;160 days respectively (p &lt; 0.001). Combined treatment with bevacizumab and mithramycin A produced synergistic tumor suppression, which was consistent with suppression of the expression of Sp1 and its downstream target genes. Conclusion: Treatment with bevacizumab may block VEGF function but activate the pathway of its expression via positive feedback. Given the fact that Sp1 is an important regulator of the expression of multiple angiogenic factors, bevacizumab-initiated upregulation of Sp1 and subsequent overexpression of its downstream target genes may affect the potential angiogenic phenotype and effectiveness of antiangiogenic strategies for human carcinoid. No significant financial relationships to disclose.