EGFR-expressing Cell Lines Research Articles

Abstract 2087: Pan-HER inhibitor, varlitinib, disrupts HER/ERK signaling and causes apoptosis in triple-negative breast cancer cells

Abstract Background: Triple-negative breast cancer (TNBC), characterized by aggressive behavior and poor prognosis, represents an important clinical challenge because there is no well-established target therapy. Therefore, the identification and validation of a targeted therapy for TNBC is an urgent need. Molecular profiling studies have shown some TNBC tumors harboring aberrant epidermal growth factor receptor (EGFR) or human epidermal growth factor receptor (HER) signaling, suggesting therapeutic potential with EGFR inhibitors. Varlitinib (ASLAN001) is a small molecule reversible pan-HER inhibitor of EGFR (HER1), HER2 and HER4. To-date, varlitinib has been extensively investigated in several tumor types, including HER2 positive metastatic breast cancer. TNBC is known to demonstrate expression of EGFR. Since varlitinib also targets EGFR signaling, we hypothesized that it may also have antitumor efficacy in TNBC. Methods: MDA-MB-231 and MDA-MB-468 TNBC cell lines were used for in vitro studies. Cell viability was examined by MTT assay. Apoptotic effects were examined by flow cytometry and Western blot. Signal transduction pathways in cells were assessed by Western blot. Results: We first examined the protein expression of EGFR in a panel of TNBC cell lines. We identified MDA-MB-468 and MDA-MB-231 as EGFR-expressing cell lines. We found that varlitinib significantly inhibited cell viability and induced cell apoptosis in MDA-MB-468 cells but not in MDA-MB-231 cells. MDA-MB-231 cells carry the KRAS G13D mutation that drives downstream ERK signaling. We therefore examined the downstream signaling proteins of EGFR, including PI3K/Akt and MAPK/ERK signaling. Results showed that the protein levels of p-MEK and p-ERK were decreased in varlitinib-sensitive MDA-MB-468 cell lines, but there was no significant change in these phospho-proteins in varlitinib-resistant MDA-MB-231 cells. Furthermore, we found that ERK inhibition resensitized varlitinib-resistant cells to varlitinib-induced cell death. In addition, ectopic expression of ERK reduced the varlitinib-induced apoptosis on varlitinib-sensitive MDA-MB-468 cells. In addition, MDA-MB-468 cells are known to harbor p53-R273H gain-of-function mutation that may activate EGFR-signaling, whether the mutation is associated with varlitinib sensitivity needs further investigation. Conclusions: In this study, we identified TNBC as another tumor type that may be sensitive to varlitinib’s antitumor activity through the inhibition of HER/MAPK signaling and subsequent increase in apoptotic activity. Citation Format: Chun-Yu Liu, Tzu-Ting Huang, Chun-Teng Huang, Hsiu-Ping Yang, Ling-Ming Tseng, Chung-Wai Shiau, Kuen-Feng Chen. Pan-HER inhibitor, varlitinib, disrupts HER/ERK signaling and causes apoptosis in triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2087. doi:10.1158/1538-7445.AM2017-2087

Anti-EGFR monoclonal antibodies enhance sensitivity to DNA-damaging agents in BRCA1-mutated and PTEN-wild-type triple-negative breast cancer cells.

Increased epidermal growth factor receptor (EGFR) expression in triple-negative breast cancer (TNBC) is recognized as a promising therapeutic target, specifically through the use of selective EGFR inhibitors combined with chemotherapies. TNBC is characterized by genetic instability that leads to increased sensitivity to cytotoxic agents. We analyzed the effect of anti-EGFR monoclonal antibodies (mAbs; cetuximab and panitumumab) in combination with chemotherapeutic agents (docetaxel, cisplatin, and epirubicin) on EGFR-expressing TNBC cell lines that have different mutation statuses for one oncogene (KRAS) and two tumor suppressor genes (PTEN and BRCA1). Both mAbs failed to improve the cytotoxic effect of chemotherapies in the KRAS mutant cell line (MDA-MB-231) and PTEN-null cell lines (HCC-1937 and MDA-MB-468). In contrast, mAbs combined with DNA-damaging agents (cisplatin or epirubicin) had a synergistic effect in the BRCA1-mutant cell line SUM-1315 (wild-type KRAS and PTEN). The reintroduction of wild-type BRCA1 into SUM-1315 cells abolished this synergism. The improved effect of combination therapy was associated with cell cycle arrest at G1 phase and inhibition of the phosphorylation of EGFR and ERK1/2 proteins. These results suggest that patients with BRCA1-associated TNBC without genetic alterations in the PTEN and KRAS genes may have improved therapeutic responses to anti-EGFR mAbs combined with DNA-damaging agents. © 2017 Wiley Periodicals, Inc.

Abstract B068: Hydrocortisone, prednisolone, methylprednisolone, and dexamethasone inhibit CetuGEX™-mediated ADCC in vitro

Abstract Introduction: Antibody dependent cellular cytotoxicity is a key mode of action of therapeutic antibodies. ADCC enhancement as well as a broader coverage of F/V allotypes leads to maximal utilization of the anti-tumor effect. Glycooptimization provides the best available opportunity to maximize ADCC activity of monoclonal antibodies. Utilizing the GEX™ platform allows the combination of minimizing fucosylation, maximizing galactosylation and branching, which maximally increases the ADCC activity. The GEX™ platform comprises a comprehensive portfolio of proprietary glycoengineered human suspension cell lines, which allow for the high yield production of proteins with tailored glycosylation patterns. Productivities of 22g/L perfusion bioreactor volume have been achieved for ADCC optimized monoclonal antibodies. Cancer therapies with monoclonal antibodies including CetuGEX™ are often accompanied by treatment with glucocorticoids in order to prevent or counteract side effects like infusion reactions. However, the immunosuppressive character of glucocorticoids may also have the potential to inhibit the ADCC effect and immune cells mediating ADCC, respectively. In this study, we analyzed the influence of different glucocorticoids on ADCC mediated by CetuGEX™. Methods: By using a europium release assay with an EGFR-expressing cancer cell line and PBMCs as effector cells we tested the ability of hydrocortisone, prednisolone, methylprednisolone and dexamethasone to inhibit ADCC mediated by CetuGEX™. In addition, we analyzed the direct effect of glucocorticoids on effector and target cells using flow cytometry. Results: All tested glucocorticoids inhibited the CetuGEX™-mediated ADCC. This effect was not caused by a decreased expression of EGFR on the target cells. NK cells which are key effector cells mediating ADCC were found to be less activated in presence of glucocorticoids and CetuGEX™ compared to CetuGEX™ alone. Conclusions: Glucocorticoids have a negative influence on ADCC mediated by NK cells which is one of the key modes of actions of CetuGEX™ and other ADCC enhanced antibodies. Therefore, the use of glucocorticoids during may impair clinical the efficacy ADCC-enhanced antibodies and should be avoided or at least minimized. Citation Format: Christoph Goletz, Johanna Rühmann, Beate Habel, Antje Danielczyk, Steffen Goletz. Hydrocortisone, prednisolone, methylprednisolone, and dexamethasone inhibit CetuGEX™-mediated ADCC in vitro [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B068.

Anti-Epidermal Growth Factor Receptor Gene Therapy for Glioblastoma.

Glioblastoma multiforme (GBM) is the most common and aggressive primary intracranial brain tumor in adults with a mean survival of 14 to 15 months. Aberrant activation of the epidermal growth factor receptor (EGFR) plays a significant role in GBM progression, with amplification or overexpression of EGFR in 60% of GBM tumors. To target EGFR expressed by GBM, we have developed a strategy to deliver the coding sequence for cetuximab, an anti-EGFR antibody, directly to the CNS using an adeno-associated virus serotype rh.10 gene transfer vector. The data demonstrates that single, local delivery of an anti-EGFR antibody by an AAVrh.10 vector coding for cetuximab (AAVrh.10Cetmab) reduces GBM tumor growth and increases survival in xenograft mouse models of a human GBM EGFR-expressing cell line and patient-derived GBM. AAVrh10.CetMab-treated mice displayed a reduction in cachexia, a significant decrease in tumor volume and a prolonged survival following therapy. Adeno-associated-directed delivery of a gene encoding a therapeutic anti-EGFR monoclonal antibody may be an effective strategy to treat GBM.

Anti-EGFR monoclonal antibodies and EGFR tyrosine kinase inhibitors as combination therapy for triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is characterized by overexpression of epidermal growth factor receptor (EGFR) and activation of its downstream signaling pathways. Dual targeting of EGFR using one monoclonal antibody (mAb; cetuximab or panitumumab) and one tyrosine kinase inhibitor (EGFR-TKI; gefitinib or erlotinib) is a potential therapeutic approach. We investigated the effect of these therapies in EGFR-expressing TNBC cell lines that do or do not harbor the main activating mutations of EGFR pathways. Cell lines were sensitive to EGFR-TKIs, whereas mAbs were active only in MDA-MB-468 (EGFR amplification) and SUM-1315 (KRAS and PTEN wild-type) cells. MDA-MB-231 (KRAS mutated) and HCC-1937 (PTEN deletion) cells were resistant to mAbs. The combined treatment resulted in a synergistic effect on cell proliferation and superior inhibition of the RAS/MAPK signaling pathway in mAb-sensitive cells. The anti-proliferative effect was associated with G1 cell cycle arrest followed by apoptosis. Sensitivity to therapies was characterized by induction of positive regulators and inactivation of negative regulators of cell cycle. These results suggest that dual EGFR inhibition might result in an enhanced antitumor effect in a subgroup of TNBC. The status of EGFR, KRAS and PTEN could be used as a molecular marker for predicting the response to this therapeutic strategy.

Abstract 4212: Molecular imaging of EGFR-expressing tumors with novel targeted protein scaffold, anti-EGFR repebody

Abstract Repebody is a binding scaffold based on variable lymphocyte receptors which are nonimmunoglobulin antibodies composed of leucine-rich repeat modules in jawless vertebrates. Repebody can be developed against variety of epitopes by module engineering. In this study, EGFR-specific repebody (RBEGFR) was developed to visualize the status of receptor expression and to prevent ligand binding that may inhibit autophosphorylation and downstream intracellular signaling. We developed RBEGFR by phage display. H1650, HCC827, A549 (human non-small cell lung cancer) and HT29 (human colon cancer) were selected as EGFR expressing cell lines. MDA-MB-435 (human melanoma) and SW620 (human colon cancer) was selected as a negative control. Specific binding of RBEGFR to cells and cancer tissue was determined by immunofluorescence (IF) staining and/or FACS analysis. In vivo imaging was done by i.v. injection of FNR-675 labeled RBEGFR (30 μg/mouse) or 64Cu-NOTA- RBEGFR (7.4 MBq/mouse) in H1650, HCC827 and HT29-bearing mouse models using cooled CCD camera or microPET, respectively. Orthotopic colon cancer mice were generated by i.p. injection of Azoxymethane (AOM; 10 mg/kg) and oral administration of 2% dextran sulfate sodium (DSS). In vivo imaging was done by i.v. injection FNR-675 labeled RBEGFR (30 μg/mouse) in AOM/DSS mouse models using cooled CCD camera or fluorescence microendoscopy. In vitro and in vivo IF staining demonstrated that strong binding of RBEGFR to H1650, HCC827 and HT29, but not to MDA-MB-435 and SW620. In vivo near infrared (NIR) imaging demonstrated specific targeting of FNR-675-RBEGFR to grafted H1650, HCC827 and HT29 tumor in mice. The 64Cu-NOTA- RBEGFR was detected at the implanted tumor from 1 h (SUVmax: 1.34±0.12) after the injection, peaked at 6 h (1.75±0.18), maintained to 24 h (1.33±0.17). The radioactivity significantly decreased by blocking with cold form of 50 μM naïve RBEGFR 1 day before injection of 64Cu-NOTA- RBEGFR, indicating specific binding of RBEGFR to EGFR in vivo. Optical NIR imaging after i.v. injection of FNR-675-RBEGFR showed specific signal in the abdomen of AOM/DSS mice, but not in control mice. Correlation with surgical/necropsy imaging, fluorescence endoscopy and pathology revealed strong accumulation of FNR-675-RBEGFR in malignant dysplasia, but weak or no accumulation in low grade tumor or benign lesion. In conclusion, the RBEGFR could be developed for specific targeting of cancer overexpressing EGFR. The fluorescence-labeled RBEGFR could be developed for imaging agent for detecting colonic dysplasia and assessing EGFR status. In particular, this agent may have a potential as an imaging companion diagnostics to predict therapeutic outcome of targeted therapy with monoclonal antibody for EGFR through pre-therapeutic visualization of EGFR status. Our work provides a basis to develop potential strategy of targeted immune-detection of cancers which may replace monoclonal antibodies. Citation Format: Misun Yun, Dong-Yeon Kim, Hyeon-Sik Kim, Jin Hai Zheng, AYoung Pyo, Jung-Joon Min. Molecular imaging of EGFR-expressing tumors with novel targeted protein scaffold, anti-EGFR repebody. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4212.

Abstract 2123: Antibody-dependent cell-mediated cytotoxicity (ADCC) tolerance to monoclonal antibody therapeutics in human breast and colon cancer cell lines

Abstract Resistance to monoclonal antibody (mAb) therapy limits clinical utility. ADCC is a significant mechanism of action of mAb-elicited tumor response. Postulated mechanisms of mAb resistance include, alteration in cell signaling pathways (PI3K/Akt/Ras-MAPK signaling), over activation/expression of alternate receptor kinases (c-Met/ IGF-1R), or proteolysis of extracellular domains harboring target epitopes. However, most studies on mAb resistance have utilized cancer cells conditioned with mAbs alone, in absence of immune effector cells. We developed an in vitro selection model wherein human HER2 positive breast cancer cell lines (BT474, SKBR3) and EGFR-expressing colorectal cancer cell lines (HT29, DLD1) were subjected to acute ADCC (>90% cell death) with high effector-target (100:1) ratio repeatedly with trastuzumab and cetuximab, respectively, using human peripheral blood mononuclear cells (PBMCs) as effectors. Surviving cells were allowed to recover to confluence over 8-10 weeks for 10 total rounds of ADCC selection. Internal controls included mock-treated parental, isotype-IgG1, effector cells only, and mAb alone. In vitro ADCC assays based on calcein fluorescent labeling of live target cells, demonstrated significant reduction (20%, p<0.005) in cell lysis in immune-selected cell lines compared to parental controls. ADCC-conditioned HT29 cells demonstrated 5-fold reduction in EGFR gene copy number by fluorescence in situ hybridization (FISH, p<0.0001), 2-fold reduction in mRNA level (qPCR, p< 0.01) and 1.8-fold reduction in protein level (immunoblot densitometry, p<0.01). In BT474 breast cells, ADCC-conditioned cells acquired elongated fibroblast-like morphology. Transcriptome-wide next-generation RNA sequencing (Illumina NextSeq 500, 2 × 150 bp paired-end, with over 100 million paired-end reads/sample), coupled with bioinformatic analyses (Reactome pathway database) revealed gene expression changes in immune-selected cells. These involve changes in key immune signaling pathways such as NK-cell and T-cell maturation, class I MHC antigen processing, and immune regulatory pathways, among others. Further validation through q-PCR analyses and loss/ gain of expression is ongoing. Future investigation will also determine whether mAb combination with agonist CD137 mAb (co-stimulatory NK receptor) or Fc-engineered mAbs (e.g. afucosylated) will re-sensitize resistant cells. Our data indicate immune-selection by effector cells contributes to ADCC resistance in vitro. Such investigation may help to elucidate potentially targetable pathways/ markers that emerge from immune-selection with mAbs and effector cells. Citation Format: Tanuka Biswas, Rebecca Fritzemeier, Adam Mark, Tobias Meißner, Brandon M. Young, Mark D. Pegram. Antibody-dependent cell-mediated cytotoxicity (ADCC) tolerance to monoclonal antibody therapeutics in human breast and colon cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2123.

Abstract 593: Highly cytotoxic EGFR/CD16A TandAbs specifically recruit NK cells to potently kill various types of solid tumors

The effect on antigenicity of covalent attachment of lipid groups to a protein antigen was investigated. Coupling of palmitic acid to ovalbumin (OVA) enhanced major histocompatibility complex (MHC) class II-restricted presentation to most OVA-specific murine T-cell clones in vitro. The enhanced antigenicity of palmitoylated antigen was localized to the level of presentation of the synthetic peptide epitope, OVA 323-339. T-cell responses to palmitoylated antigen were more difficult to block with anti-MHC class II antibodies than responses to native antigen. However, T-cell proliferation to palmitoyl (p)-OVA and native (n)-OVA were blocked equally by anti-CD4 antibodies. Taken together, the results suggest that lipid conjugation of a protein antigen leads to the formation of a lipopeptide T-cell epitope with increased affinity of binding to MHC class II and/or T-cell receptor (TcR). These results have implications for the design of synthetic peptide vaccines.

Moderate and strong static magnetic fields directly affect EGFR kinase domain orientation to inhibit cancer cell proliferation.

Static magnetic fields (SMFs) can affect cell proliferation in a cell-type and intensity-dependent way but the mechanism remains unclear. At the same time, although the diamagnetic anisotropy of proteins has been proposed decades ago, the behavior of isolated proteins in magnetic fields has not been directly observed. Here we show that SMFs can affect isolated proteins at the single molecular level in an intensity-dependent manner. We found that Epidermal Growth Factor Receptor (EGFR), a protein that is overexpressed and highly activated in multiple cancers, can be directly inhibited by SMFs. Using Liquid-phase Scanning Tunneling Microscopy (STM) to examine pure EGFR kinase domain proteins at the single molecule level in solution, we observed orientation changes of these proteins in response to SMFs. This may interrupt inter-molecular interactions between EGFR monomers, which are critical for their activation. In molecular dynamics (MD) simulations, 1-9T SMFs caused increased probability of EGFR in parallel with the magnetic field direction in an intensity-dependent manner. A superconducting ultrastrong 9T magnet reduced proliferation of CHO-EGFR cells (Chinese Hamster Ovary cells with EGFR overexpression) and EGFR-expressing cancer cell lines by ~35%, but minimally affected CHO cells. We predict that similar effects of magnetic fields can also be applied to some other proteins such as ion channels. Our paper will help clarify some dilemmas in this field and encourage further investigations in order to achieve a better understanding of the biological effects of SMFs.

Abstract 2596: ACP-196: a novel covalent Bruton's tyrosine kinase (Btk) inhibitor with improved selectivity and in vivo target coverage in chronic lymphocytic leukemia (CLL) patients

Abstract Ibrutinib, a first generation Btk inhibitor, is approved for the treatment of CLL and mantle cell lymphoma; known toxicities include atrial fibrillation, diarrhea, rash, arthralgia and bleeding events (1). Recent reports show ibrutinib's off target effects may negatively impact its potential for combined therapy with anti-CD20 antibodies (2,3). Here we describe the pharmacologic characterization of ACP-196 a potent, novel second generation Btk inhibitor, which binds covalently to Cys481 with improved selectivity and in vivo target coverage. Compared to ibrutinib and CC-292, ACP-196 demonstrated higher selectivity for Btk when profiled against a panel of 395 non-mutant kinases (1 μM) in a competitive binding assay. IC50 determinations on 9 kinases with a Cys in the same position as Btk showed ACP-196 to be the most selective. The improved selectivity is related to the reduced intrinsic reactivity of ACP-196's electrophile. Importantly, unlike ibrutinib, ACP-196 did not inhibit EGFR, Itk or Txk. Phosphoflow assays on EGFR expressing cell lines confirmed ibrutinib's EGFR inhibition (EC50: 47-66 nM) with no inhibition observed for ACP-196 at 10 μM. These data may explain the ibrutinib-related incidence of diarrhea and rash. Ibrutinib's potency on Itk and Txk may explain why it interferes with cell-mediated anti-tumor activities of therapeutic CD20 antibodies and immune-mediated killing in the tumor microenvironment (2,3). In human whole blood, ACP-196 and ibrutinib showed robust and equipotent inhibitory activity on B-cell receptor induced responses in the low nM range, whereas CC-292 was 10-20 fold less potent. In vivo, oral administration of ACP-196 in mice resulted in dose-dependent inhibition of anti-IgM-induced CD86 expression in CD19+ splenocytes with an ED50 of 0.34 mg/kg compared to 0.91 mg/kg for ibrutinib. A similar model was used to compare the duration of Btk inhibition after a single oral dose of 25 mg/kg. ACP-196 and ibrutinib inhibited CD86 expression >90% at 3h and ∼50% at 24h postdose. In contrast, CC-292 inhibited ∼50% at 3h and ∼20% at 24h postdose. An ELISA based Btk target occupancy assay was developed to measure target coverage in preclinical and clinical studies. In healthy volunteers, ACP-196 at an oral dose of 100 mg QD showed >90% target coverage over a 24h period. Btk occupancy and regulation of the PD markers (CD69 and CD86) correlated with PK parameters for exposure. In CLL patients, after 7 days of dosing with ACP-196 at 200 mg QD, 94% Btk target occupancy was observed compared with ∼80% reported for ibrutinib at 420 mg QD (4). In conclusion, ACP-196 is a novel Btk inhibitor with key pharmacologic differentiators versus ibrutinib and CC-292. ACP-196 is currently being evaluated in clinical trials. 1. IMBRUVICA package insert 2014 2. Rajasekaran Blood 2014 Abstr # 3118 3. Da Roit Haematologica 2014 4. Byrd NEJM 2013 Citation Format: Todd Covey, Tjeerd Barf, Michael Gulrajani, Fanny Krantz, Bart van Lith, Elena Bibikova, Bas van de Kar, Edwin de Zwart, Ahmed Hamdy, Raquel Izumi, Allard Kaptein. ACP-196: a novel covalent Bruton's tyrosine kinase (Btk) inhibitor with improved selectivity and in vivo target coverage in chronic lymphocytic leukemia (CLL) patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2596. doi:10.1158/1538-7445.AM2015-2596

Abstract 652: Antibody-drug conjugates (ADCs) of indolino-benzodiazepine DNA-alkylating agents

Abstract There are over thirty ADCs in the clinic today, reflecting the interest in ADCs for the treatment of cancer. Most of these ADCs utilize a tubulin-interacting small molecule as their cytotoxic payload. In order to continue to extend the utility of ADCs to more types of cancers, there is a strong interest in developing cytotoxic payloads with other mechanisms of action. Previously we have described the development of a class of indolino-benzodiazepine dimers (IGNs) with high potency and specificity. ADCs made with a mono-imine containing IGN, which only alkylate DNA, were found to display improved tolerability, along with the absence of delayed toxicity, compared to their DNA-cross-linking di-imine counterparts. Additional structure-activity relationship studies with mono-imine containing IGNs have resulted in two promising new leads, IGN-D1 and IGN-P1, which are conjugated to an antibody via either a disulfide or peptide linker, respectively. ADCs of IGN-D1 and IGN-P1 were prepared for evaluation using a folate receptor α (FRα)-binding antibody, anti-FRα, and EGFR-binding antibody, anti-EGFR. Anti-FRα-IGN-D1 and anti-FRα-IGN-P1 were found to be highly potent against the FRα-expressing KB cell line (IC50 values of 8 pM and 4 pM, respectively) and also demonstrated strong activity against nearby target-negative cancer cells. The addition of excess unconjugated antibody blocked the cytotoxic effect of these conjugates, thus demonstrating that their activity is antigen specific. In addition, both anti-FRα-IGN conjugates were found to be highly potent (IC50 values of 100 pM and 30 pM, respectively) against the breast ductal carcinoma cell line, T47D, despite only a moderate level of antigen expression on the target cells. Similarly, anti-EGFR-IGN-D1 and anti-EGFR-IGN-P1 were found to have IC50 values in the picomolar range against a panel of EGFR-expressing cell lines. In vivo, anti-FRα-IGN-D1 and anti-FRα-IGN-P1 were found to be effective against NCI-H2110 non-small cell lung cancer xenografts, with anti-tumor activity observed at single doses as low as 3 μg/kg (payload dose, equivalent to 0.15 mg/kg Ab dose). These data support evaluation of new ADC therapies with these two new IGNs for solid tumor indications as well as tumors with low antigen expression. Citation Format: Michael L. Miller, Manami Shizuka, Nathan Fishkin, Emily Reid, Katie Archer, Erin Maloney, Chen Bai, Olga Ab, Nick C. Yoder, Rui Wu, Erica Hong, Megan Bogalhas, Alan Wilhelm, Kathleen Whiteman, Ravi Chari. Antibody-drug conjugates (ADCs) of indolino-benzodiazepine DNA-alkylating agents. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 652. doi:10.1158/1538-7445.AM2015-652

Abstract 1669: Preclinical efficacy of Sym004, novel anti-EGFR antibody, in esophageal squamous cell carcinoma cell lines

Abstract Background: Epidermal growth factor receptor (EGFR) is a well-validated oncology target molecule for monoclonal antibody therapies. Sym004 is a novel anti-EGFR antibody mixture that consists of two recombinant IgG1 antibodies against domain III of the EGFR. The primary mechanism of action of Sym004 is thought to be EGFR cross-linking, internalization and degradation of the EGFR from the cell surface. EGFR is highly expressed in the overwhelming majority of esophageal squamous cell carcinoma (ESCC). We, therefore, investigated the effect of Sym004 in human ESCC cell lines in the present study. Method: In this preclinical study, approximately 50 ESCC cell lines and 3 kinds of anti-EGFR antibodies (Sym004, cetuximab, and panitumumab) were used. According to protein expression status and gene mutation status analyzed by Western blotting and next generation sequencing, these ESCC cell lines were classified into several groups. High EGFR-expressing cell lines were used for further investigations. Internalization of anti-EGFR antibodies into ESCC cells and inhibition of the EGFR signaling cascade by anti-EGFR antibodies were studied in in vitro experiments. Growth inhibitions by anti-EGFR antibody treatment were investigated in in vitro and in vivo experiments. The present preclinical studies were conducted at the National Cancer Center East Japan as collaborative studies with Merck Serono. Result: Even though protein expression of EGFR was detected in all ESCC cell lines, a variety of the expression levels were seen among the cell lines consistent with the previous data of clinical specimens. In addition, following gene alterations were found: EGFR amplification, Ras mutation, and PIK3CA mutation, and their frequency was 17% (8/48), 6.3% (3/48) and 8.3% (4/48), respectively. More effective internalization of Sym004 was observed than cetuximab and panitumumab in the high EGFR-expression cell lines. In a growth inhibition assay, more potent anti-proliferative activity of Sym004 was observed than those of other anti-EGFR antibodies in some cell lines. No obvious correlation between EGFR expression and the anti-proliferative activity was observed, although cell lines with higher EGFR expression showed a trend toward higher sensitivity to Sym004. In vivo xenograft studies of Sym004 using human ESCC cell lines are currently underway and will be presented in the annual meeting. Conclusions: These studies showed that Sym004 exhibited antitumor activity in some ESCC cell lines in preclinical settings, and are expected to provide a scientific rationale along with a possible clinical indication of ESCC in clinical setting. Citation Format: Shota Fukuoka, Takashi Kojima, Yoshikatsu Koga, Mayumi Yamauchi, Rie Komatsuzaki, Kazuhiko Aoyagi, Hiroki Sasaki, Masahiro Yasunaga, Yasuhiro Matsumura, Toshio Kuronita, Toshihiko Doi, Atsushi Ohtsu. Preclinical efficacy of Sym004, novel anti-EGFR antibody, in esophageal squamous cell carcinoma cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1669. doi:10.1158/1538-7445.AM2015-1669

EGFR over-expression in non-small cell lung cancers harboring EGFR mutations is associated with marked down-regulation of CD82

Epidermal growth factor receptor (EGFR) gene mutations are strongly associated with lung adenocarcinoma and favorable response to EGFR tyrosine kinase inhibitor. The mutated EGFR proteins (EGFRs) are hyper-phosphorylated and refractory to receptor down-regulation. To address the discrepancy between hyper-phosphorylation and lack of down-regulation of mutant EGFRs, we have examined the expression of EGFR negative regulators in non-small cell lung cancer (NSCLC) cell lines. We found that NSCLC cell lines expressing mutant EGFRs often had low expression of various negative regulators for EGFR. Among them, tumor suppressor CD82 was up-regulated by wild type (WT) EGFR but down-regulated by mutant EGFRs. Reconstitution of CD82 exerted stronger suppressive effects on mutant EGFRs than on WT EGFR. Active exportation of CD82 through the exosome was one of the mechanisms involved in achieving the overall CD82 down-regulation in mutant EGFR-expressing lung cancer cell lines. Over-expression of mutant EGFR protein frequently occurred in the lung cancer tissues of mutant EGFR-transgenic mice and also associated with CD82 down-regulation. Immunoblot analyses on the tumor tissues from 23 lung adenocarcinoma patients (12 with WT EGFR, and 11 with mutant EGFRs) also identified significantly stronger down-regulation of CD82 in tumors with mutant EGFRs than WT. Our data indicate that CD82 down-regulation could be a critical step involved in the EGFR over-expression and the stronger tumorigenic activity triggered by EGFR mutations. Up-regulation of the CD82 level may become a promising new treatment strategy for lung adenocarcinoma.

Shikonin and its derivatives inhibit the epidermal growth factor receptor signaling and synergistically kill glioblastoma cells in combination with erlotinib.

Overexpression and mutation of the epidermal growth factor receptor (EGFR) gene play a causal role in tumorigenesis and resistance to treatment of glioblastoma (GBM). EGFR inhibitors such as erlotinib are currently used for the treatment of GBM; however, their efficacy has been limited due to drug resistance. New treatment strategies are therefore urgently needed. Shikonin, a natural naphthoquinone, induces both apoptosis and necroptosis in human glioma cells, but the effectiveness of erlotinib-shikonin combination treatment as well as the underlying molecular mechanisms is unknown yet. In this study, we investigated erlotinib in combination with shikonin and 14 shikonin derivatives in parental U87MG and transfected U87MG.ΔEGFR GBM cells. Most of the shikonin derivatives revealed strong cytotoxicity. Shikonin together with five other derivatives, namely deoxyshikonin, isobutyrylshikonin, acetylshikonin, β,β-dimethylacrylshikonin and acetylalkannin showed synergistic cytotoxicity toward U87MG.ΔEGFR in combination with erlotinib. Moreover, the combined cytotoxic effect of shikonin and erlotinib was further confirmed with another three EGFR-expressing cell lines, BS153, A431 and DK-MG. Shikonin not only dose-dependently inhibited EGFR phosphorylation and decreased phosphorylation of EGFR downstream molecules, including AKT, P44/42MAPK and PLCγ1, but also together with erlotinib synergistically inhibited ΔEGFR phosphorylation in U87MG.ΔEGFR cells as determined by Loewe additivity and Bliss independence drug interaction models. These results suggest that the combination of erlotinib with shikonin or its derivatives might be a potential strategy to overcome drug resistance to erlotinib.

Design of an EGFR-targeting toxin for photochemical delivery: in vitro and in vivo selectivity and efficacy.

The number of epidermal growth factor receptor (EGFR)-targeting drugs in the development for cancer treatment is continuously increasing. Currently used EGFR-targeted monoclonal antibodies and tyrosine kinase inhibitors have specific limitations related to toxicity and development of resistance, and there is a need for alternative treatment strategies to maximize the clinical potential of EGFR as a molecular target. This study describes the design and production of a novel EGFR-targeted fusion protein, rGel/EGF, composed of the recombinant plant toxin gelonin and EGF. rGel/EGF was custom-made for administration by photochemical internalization (PCI), a clinically tested modality for cytosolic release of macromolecular therapeutics. rGel/EGF lacks efficient mechanisms for endosomal escape and is therefore minimally toxic as monotherapy. However, PCI induces selective and efficient cytosolic release of rGel/EGF in EGFR-expressing target cells by light-directed activation of photosensitizers accumulated selectively in tumor tissue. PCI of rGel/EGF was shown to be highly effective against EGFR-expressing cell lines, including head and neck squamous cell carcinoma (HNSCC) cell lines resistant to cetuximab (Erbitux). Apoptosis, necrosis and autophagy were identified as mechanisms of action following PCI of rGel/EGF in vitro. PCI of rGel/EGF was further shown as a highly tumor-specific and potent modality in vivo, with growth inhibitory effects demonstrated on A-431 squamous cell carcinoma (SCC) xenografts and reduction of tumor perfusion and necrosis induction in SCC-026 HNSCC tumors. Considering the small amount of rGel/EGF injected per animal (0.1 mg/kg), the presented in vivo results are highly promising and warrant optimization and production of rGel/EGF for further preclinical evaluation with PCI.

Evaluation of Targets for Maytansinoid ADC Therapy Using a Novel Radiochemical Assay.

Many antibody-drug conjugates (ADCs) become active only after antigen-mediated internalization and release of the cytotoxic agent via antibody degradation. Quantifying these processes can provide critical information on the suitability of a particular receptor target or antibody for ADC therapy by providing insight into the amount of cytotoxic agent released. We describe a simple and inexpensive radiolabel assay to monitor this process in cultured cancer cells. Monoclonal antibodies were trace-labeled at their lysine residues by treatment with the N-hydroxysuccinimide ester of [(3)H]propionic acid. Human cancer cell cultures were treated with the labeled antibody at concentrations sufficient to saturate the targeted antigen. After washing to remove unbound antibody, cells were incubated and analyzed for antigen expression, conjugate degradation and catabolite formation. Results were compared with data obtained from similar assays run with radiolabeled antibody-[(3)H]maytansinoid conjugates ([(3)H]AMCs). To exemplify the method, studies were conducted with a panel of [(3)H]propionamide-antibodies to evaluate processing efficiency in EGFR-expressing SCCHN cell lines, and in NHL cell lines expressing the B-cell targets CD19, CD20, CD22 and CD37. Use of the [(3)H]propionamide-antibody assay yielded cell-mediated processing results similar to those obtained with corresponding maytansinoid ADCs. Further exploration allowed comparison of expression levels, antigen-dependent degradation, and catabolite formation across a panel of EGFR-expressing SCCHN cell lines, and for multiple targets in various B-cell cancer indications. The [(3)H]propionamide-antibody assay described here is a sensitive, facile method which enables rapid and robust assessment of relative antibody processing amounts for target, antibody, and cell line evaluation.

JAK3 inhibitor VI is a mutant specific inhibitor for epidermal growth factor receptor with the gatekeeper mutation T790M.

To identify non-quinazoline kinase inhibitors effective against drug resistant mutants of epidermal growth factor receptor (EGFR). A kinase inhibitor library was subjected to screening for specific inhibition pertaining to the in vitro kinase activation of EGFR with the gatekeeper mutation T790M, which is resistant to small molecular weight tyrosine kinase inhibitors (TKIs) for EGFR in non-small cell lung cancers (NSCLCs). This inhibitory effect was confirmed by measuring autophosphorylation of EGFR T790M/L858R in NCI-H1975 cells, an NSCLC cell line harboring the gatekeeper mutation. The effects of a candidate compound, Janus kinase 3 (JAK3) inhibitor VI, on cell proliferation were evaluated using the MTT assay and were compared between T790M-positive and -negative lung cancer cell lines. JAK3 inhibitor VI was modeled into the ATP-binding pocket of EGFR T790M/L858R. Potential physical interactions between the compound and kinase domains of wild-type (WT) or mutant EGFRs or JAK3 were estimated by calculating binding energy. The gatekeeper residues of EGFRs and JAKs were aligned to discuss the similarities among EGFR T790M and JAKs. We found that JAK3 inhibitor VI, a known inhibitor for JAK3 tyrosine kinase, selectively inhibits EGFR T790M/L858R, but has weaker inhibitory effects on the WT EGFR in vitro. JAK3 inhibitor VI also specifically reduced autophosphorylation of EGFR T790M/L858R in NCI-H1975 cells upon EGF stimulation, but did not show the inhibitory effect on WT EGFR in A431 cells. Furthermore, JAK3 inhibitor VI suppressed the proliferation of NCI-H1975 cells, but showed limited inhibitory effects on the WT EGFR-expressing cell lines A431 and A549. A docking simulation between JAK3 inhibitor VI and the ATP-binding pocket of EGFR T790M/L858R predicted a potential binding status with hydrogen bonds. Estimated binding energy of JAK3 inhibitor VI to EGFR T790M/L858R was more stable than its binding energy to the WT EGFR. Amino acid sequence alignments revealed that the gatekeeper residues of JAK family kinases are methionine in WT, similar to EGFR T790M, suggesting that TKIs for JAKs may also be effective for EGFR T790M. Our findings demonstrate that JAK3 inhibitor VI is a gatekeeper mutant selective TKI and offer a strategy to search for new EGFR T790M inhibitors.

Abstract 3680: (R,R')-4′-methoxy-1-naphtylfenoterol inhibits pro-survival signaling, proliferation and motility of select human melanoma cell lines

Abstract (R,R')-4′-methoxy-1-naphtylfenoterol (MNF) inhibits proliferation of 1321N1 astrocytoma cell line in a β2-adrenergic receptor (β2AR)-dependent manner, while reducing rat C6 glioma growth in a xenograft mouse model. MNF interferes also with pro-oncogenic signaling induced by the orphan G-protein-linked receptor, GPR55, in human HepG2 hepatocarcinoma and PANC-1 pancreatic carcinoma cells. Here, we utilized a number of cellular and biochemical techniques to investigate the anti-tumorigenic potential of MNF in a panel of human melanoma cell lines. Cell migration, which was performed using scratch wound healing assay, demonstrated the ability of MNF to dose-dependently reduce the motility of EGFR-expressing UACC-647 and M93-047 cell lines in a biphasic (IC50 = 0.32 nM and 1.48 µM) and sigmoidal (IC50 = 7.14 nM) fashion, respectively. In contrast, EGFR-negative UACC-903 cells were unresponsive to MNF. The proliferation rate of UACC-647 cells was diminished by 38 ± 3.7% in the presence of MNF, with an IC50 of 136 nM. Pharmacological inhibition of β2AR with the selective antagonist ICI-118,551 (50 nM) did not confer protection against the actions of MNF on cell motility and proliferation. U-shaped dose-response curves, with a nadir at 1 nM, were observed when the levels of phosphorylated MEK and ERK were determined in MNF-treated UACC-647 cells. A similar dose-response relationship was observed with the β2AR agonists, epinephrine and (R,R')-fenoterol, and cell pretreatment with 50 nM ICI-118,551 abolished MNF-evoked drop in phospho-ERK levels. However, MNF markedly increased inhibitory phosphorylation of eukaryotic elongation factor 2 (eEF2) on Thr56, which was accompanied by global defect in protein biosynthesis as revealed by pulse-labeling experiments with [35S]-methionine. We conclude that the anti-tumorigenic functions of MNF most likely reflect dual effects of MNF through interaction with different receptors in melanoma. This work was supported entirely by the Intramural Research Program of the National Institute on Aging/NIH and NIA/NIH contract N01-AG31009, and the Foundation for Polish Science (TEAM 2009-4/5 programme). Citation Format: Artur Wnorowski, Rajib K. Paul, Lucita Jimenez, Lawrence Toll, Abdelmohsen Kotb, Michel Bernier, Irving W. Wainer. (R,R')-4′-methoxy-1-naphtylfenoterol inhibits pro-survival signaling, proliferation and motility of select human melanoma cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3680. doi:10.1158/1538-7445.AM2014-3680

Abstract 2893: Engineered release and presentation of antibody-bound viral antigens: A highly specific and novel immunotherapeutic approach to target cancer in vivo

Abstract Harnessing the power of adaptive immunity to combat cancer has been a long-term goal of translational immunotherapy. Tumor-specific immunity, where present, is typically at low frequency and affinity with compromised effector function. By contrast, immunity against persistent herpesviruses in man is characterised by high affinity cytotoxic T-lymphocytes (CTL) at high frequency with potent effector function. Furthermore, the immunosubversive mechanisms employed by herpesviruses show striking parallels to tumors, yet the associated anti-viral immunity limits these to life-long asymptomatic infections. We reasoned that the delivery of immunodominant viral peptide epitopes to the tumor surface might facilitate passive-loading of peptides into empty MHC class-I molecules, effectively mimicking viral infection, rendering tumors susceptible to lysis by anti-viral immunity. To address this we developed a new class of targeting antibodies: APEC (Antibody Peptide Epitope Complexes) that are able to deliver an antigenic payload at the cell surface through proteolytic release of covalently-coupled peptide antigens. As a proof-of-concept we used clinically-validated antibodies cetuximab (anti-EGFR) and rituximab (anti-CD20) to develop APECs that are able to target human tumors. We screened 15 HLA-A*0201+ EGFR-expressing NCI-60 cell lines, CD20+ lymphoma cell lines, 20 primary CD20+ CLL tumor samples and four healthy B-cells against a library of 190 cetuximab-APECs (cAPEC) or rituximab (rAPEC) incorporating the immunodominant cytomegalovirus (CMV) pp65495-503 epitope and candidate protease cleavage sites following co-incubation with CMV-specific CTL (CMV-CTL). The most effective cAPEC and rAPEC were those incorporating MMP2, MMP9, Cathepsin B and Cathepsin D protease recognition domains. Very few (2/190) rAPEC were able to redirect CMV-CTL against healthy cells. Heterogeneity was observed for primary CLL tumors but a limited number of rAPEC were effective in all cases (5/190). Mechanistic studies demonstrated that: (i) peptide loading occurred at the cell surface, (ii) required the expression of target antigens at the cell surface and (iii) T-cell recognition could be inhibited by unconjugated antibody (92%) or by incubation with protease inhibitors (83%). T-cell specificity was examined using rAPEC treated tumor targets co-incubated with various HLA-matched effector T-cell populations. No activation of CD4+ was observed including CD4+CD25hi regulatory T-cell populations. Incubation with CD8+ T-cells revealed that only pp65495-503-specific CD8+ T-cells engaged with APEC-treated tumor cells. Lastly, xenograft studies using EGFR+ and CD20+ tumor cell lines demonstrated efficacy of both cAPEC and rAPEC to eliminate tumors in vivo by redirecting anti-viral CTL. These data indicate that APECs represent a powerful new approach to combat cancer. Citation Format: David G. Millar, Laura Morton, Manuela Carvalho Gaspar, Punam Mistry, Hugo De La Peña, Ricky Joseph, Sarah Penny, Oliver C. Goodyear, Margaret Goodall, Guy E. Pratt, Mark Cobbold. Engineered release and presentation of antibody-bound viral antigens: A highly specific and novel immunotherapeutic approach to target cancer in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2893. doi:10.1158/1538-7445.AM2014-2893

The effect of chelator type on in vitro receptor binding and stability in 177Lu-labeled cetuximab and panitumumab.

Monoclonal antibodies are used in the therapy of various diseases. Thanks to their high specific uptake in target tissues, these antibodies can be utilized in targeted radioimmunotherapy as carriers of radioisotopes to tumors. However, important characteristics of antibodies such as target binding and stability in the organism may be affected by various structural parameters. This study has focused on the potential influence of selected chelators on radiochemical quality and in vitro receptor binding capacity in two modified monoclonal antibodies-cetuximab and panitumumab, both ligands of the epidermal growth factor receptor (EGFR). These two antibodies were each coupled with three macrocyclic chelators (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 1,4,7-triazacyclononane-1,4,7-triacetic acid, and 3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-triene-4-(S)-(4-isothiocyanatobenzyl)-3,6,9-triacetic acid) and labeled with lutetium-177. The stability of the preparations was checked, and the cell binding to EGFR-expressing cell lines was examined. The used method led to very stable radiolabeled preparations. The results showed that binding to the target cells was not affected by the type of chelator. All three chelators may be useful for the labeling of cetuximab and panitumumab with lutetium-177 in future preclinical or clinical studies. Our study revealed previously unpublished fact that the type of chelator selected does not affect binding of EGFR-targeted antibodies labeled with lutetium-177.