EGFR-expressing Cell Research Articles

Monitoring kinetics reveals critical parameters of IgA-dependent granulocyte-mediated anti-tumor cell cytotoxicity

Human IgA antibodies effectively engage myeloid cells for the FcαRI-dependent antibody-dependent cell-mediated cytotoxicity (ADCC) of tumor cells. Established methods to investigate ADCC are the 51chromium and Calcein release assays. Their critical limitations are the end-point measurement, the unspecific release of the probes, the requirement of target cells in suspension and thus do not reflect physiologic conditions of adherently growing cells. Here we report the label-free real-time monitoring of granulocyte-mediated ADCC using an impedance-based method. We investigated the efficacy of an engineered epidermal growth factor receptor (EGFR)-directed IgA2 antibody to engage neutrophils for ADCC against a panel of adherently growing EGFR-expressing cancer cell lines majorly head and neck squamous cell carcinoma (HNSCC). The impedance assay allowed the documentation of the IgA-neutrophil-and FcαRI-signaling dependent ADCC of adherently growing target cells. While at a short-term it provided comparable results to release assays, in the long run real time monitoring also revealed cell-line specific kinetics and long-term efficacy. Although short-term results may depend on EGFR expression, long-term efficacy did not correlate with the surface level of EGFR nor of the myeloid checkpoint CD47 pointing to additional critical parameters to predict the treatment efficacy. Real-time monitoring of neutrophil-mediated ADCC allowed documenting effector cell activity and exhaustion. Along with excess expression of Mac-1 ligands, which may explain the target cell resistance, this eventually leads to tumor cell outgrowth at later time points. In conclusion, the impedance assay provides valuable information on the kinetics, effector cell performance, efficacy and critical parameters of IgA-dependent granulocyte-mediated cytotoxicity and is expected to become an important tool in its evaluation.

Exploring pitfalls of 64Cu-labeled EGFR-targeting peptide GE11 as a potential PET tracer.

The epidermal growth factor receptor (EGFR) represents an important molecular target for both radiotracer-based diagnostic imaging and radionuclide therapy of various cancer entities. For the delivery of radionuclides to the tumor, peptides hold great potential as a transport vehicle. With respect to EGFR, the peptide YHWYGYTPQNVI (GE11) has been reported to bind the receptor with high specificity and affinity. In the present study, GE11 with β-alanine (β-Ala-GE11) was conjugated to the chelating agent p-SCN-Bn-NOTA and radiolabeled with 64Cu for the first radio pharmacological evaluation as a potential probe for positron emission tomography (PET)-based cancer imaging. For better water solubility, an ethylene glycol-based linker was introduced between the peptide'sN terminus and the radionuclide chelator. The stability of the 64Cu-labeled peptide conjugate and its binding to EGFR-expressing tumor cells was investigated in vitro and in vivo, and then compared with the 64Cu-labeled EGFR-targeting antibody conjugate NOTA-cetuximab. The GE11 peptide conjugate [64Cu]Cu-NOTA-linker-β-Ala-GE11 ([64Cu]Cu-1) was stable in a buffer solution for at least 24h but only 50% of the original compound was detected after 24h of incubation in human serum. Stability could be improved by amidation of the peptide's C terminus (β-Ala-GE11-NH2 (2)). Binding assays with both conjugates, [64Cu]Cu-1 and [64Cu]Cu-2, using the EGFR-expressing tumor cell lines A431 and FaDu showed no specific binding. A pilot small animal PET investigation in FaDu tumor-bearing mice revealed only low tumor uptake (standard uptake value (SUV) < 0.2) for both conjugates. The best tumor-to-muscle ratio determined was 3.75 for [64Cu]Cu-1, at 1h post injection. In conclusion, the GE11 conjugates in its present form are not suitable for further biological investigations, since they presumably form aggregates.

Combined external beam radiotherapy with carbon ions and tumor targeting endoradiotherapy

External beam radiotherapy (EBRT) with carbon ions and endoradiotherapy using radiolabeled tumor targeting agents are emerging concepts in precision cancer therapy. We report on combination effects of these two promising strategies.Tumor targeting 131I-labelled anti-EGFR-antibody (Cetuximab) was used in the prototypic EGFR-expressing A431 human squamous cell carcinoma xenograft model. A 131I-labelled melanin-binding benzamide derivative was utilized targeting B16F10 melanoma in an orthotopic syngeneic C57bl6 model. Fractionated EBRT was performed using carbon ions in direct comparison with conventional photon irradiation.Tumor uptake of 131I-Cetuximab and 131I-Benzamide was enhanced by fractionated EBRT as determined by biodistribution studies. This effect was independent of radiation quality and significant for the small molecule 131I-Benzamide, i.e., >30% more uptake in irradiated vs. non-irradiated melanoma was found (p<0.05). Compared to each monotherapy, dual combination with 131I-Cetuximab and EBRT was most effective in inhibiting A431 tumor growth. A similar trend was seen for 131I-Benzamide and EBRT in B16F10 melanoma model. Addition of 131I-Benzamide endoradiotherapy to EBRT altered expression of genes related to DNA-repair, cell cycle and cell death. In contrast, immune-response related pathways such as type 1 interferon response genes (ISG15, MX1) were predominantly upregulated after combined 131I-Cetuximab and EBRT. The beneficial effects of combined 131I-Cetuximab and EBRT was further attributed to a reduced microvascular density (CD31) and decreased proliferation index (Ki-67).Fractionated EBRT could be favorably combined with endoradiotherapy. 131I-Benzamide endoradiotherapy accelerated EBRT induced cytotoxic effects. Activation of immune-response by carbon ions markedly enhanced anti-EGFR based endoradiotherapy suggesting further evaluation of this novel and promising radioimmunotherapy concept.

Elucidation of the critical epitope of an anti-EGFR monoclonal antibody EMab-134

The epidermal growth factor receptor (EGFR) is a type-1 transmembrane receptor tyrosine kinase, which activates the downstream signaling cascades in many tumors, such as oral and lung cancers. We previously developed EMab-134, a novel anti-EGFR monoclonal antibody (mAb), which reacts with endogenous EGFR-expressing cancer cell lines and normal cells independent of glycosylation in Western blotting, flow cytometry, and immunohistochemical analysis. EMab-134 showed very high sensitivity (94.7%) to oral squamous cell carcinomas in immunohistochemical analysis. In this study, we performed enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemical analysis to determine the epitope of EMab-134. A blocking peptide (375–394 amino acids of EGFR) neutralized the EMab-134 reaction against oral cancer cells in flow cytometry and immunohistochemistry. The minimum epitope of EMab-134 was found to be the 377-RGDSFTHTPP−386 sequence. Our findings can be applied for the production of more functional anti-EGFR mAbs that in turn can be used for antitumor treatments.

Molecularly Targeted Cancer Combination Therapy with Near-Infrared Photoimmunotherapy and Near-Infrared Photorelease with Duocarmycin-Antibody Conjugate.

Near-infrared photoimmunotherapy (NIR-PIT) is a highly selective tumor treatment that uses an antibody-photoabsorber conjugate (APC). However, the effect of NIR-PIT can be enhanced when combined with other therapies. NIR photocaging groups, based on the heptamethine cyanine scaffold, have been developed to release bioactive molecules near targets after exposure to light. Here, we investigated the combination of NIR-PIT using panitumumab-IR700 (pan-IR700) and the NIR-releasing compound, CyEt-panitumumab-duocarmycin (CyEt-Pan-Duo). Both pan-IR700 and CyEt-Pan-Duo showed specific binding to the EGFR-expressing MDAMB468 cell line in vitro In in vivo studies, additional injection of CyEt-Pan-Duo immediately after NIR light exposure resulted in high tumor accumulation and high tumor-background ratio. To evaluate the effects of combination therapy in vivo, tumor-bearing mice were separated into 4 groups: (i) control, (ii NIR-PIT, (iii) NIR-release, (iv) combination of NIR-PIT and NIR-release. Tumor growth was significantly inhibited in all treatment groups compared with the control group (P < 0.05), and significantly prolonged survival was achieved (P < 0.05 vs. control). The greatest therapeutic effect was shown with NIR-PIT and NIR-release combination therapy. In conclusion, combination therapy of NIR-PIT and NIR-release enhanced the therapeutic effects compared with either NIR-PIT or NIR-release therapy alone. Mol Cancer Ther; 17(3); 661-70. ©2017 AACR.

Amino acid starvation culture condition sensitizes EGFR-expressing cancer cell lines to gefitinib-mediated cytotoxicity by inducing atypical necroptosis.

The maintenance of the intracellular level of amino acids is crucial for cellular homeostasis. This is carried out via the regulation of both the influx from the extracellular environment and the recycling of intracellular resources. Since epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors, including gefitinib (GEF) have been reported to induce the apoptosis of several cancer cell lines, in the present study, we examined whether the cytotoxic effects of GEF are further enhanced under amino acid starvation (AAS) culture conditions. Under AAS culture conditions, the cell killing effect of GEF was synergistically pronounced in the EGFR-expressing cell lines, namely, CAL 27, Detroit 562, A549 and PANC-1 cells compared with those treated with either GEF or AAS alone. The addition of essential amino acids, but not non-essential amino acids to the cell culture medium resulted in the cancellation of this pronounced cytotoxicity. The knockdown of L-type amino acid transporter 1 (LAT-1) by siRNA also enhanced GEF-induced cytotoxicity. Therefore, the shortage of the intracellular amino acid pool appears to determine the sensitivity to GEF. Notably, this enhanced cytotoxicity is not mediated by the induction of apoptosis, but is accompanied by the pronounced induction of autophagy. The presence of necrostatin-1, an inhibitor of receptor-interacting serine/threonine-protein kinase 1 (RIPK-1), but not that of Z-VAD-fmk, attenuated the cytotoxic effects of GEF under AAS culture conditions. Electron microscopy demonstrated that the CAL 27 cells treated with GEF under AAS culture conditions exhibited swelling of the cytosol and organelles with an increased number of autophagosomes and autolysosomes, but without chromatin condensation and nuclear fragmentation. Autophagic cell death was excluded as the inhibition of autophagy did not attenuate the cytotoxicity. These results strongly suggest the induction of necroptosis in response to GEF under AAS culture conditions. However, we could not detect any phosphorylation of RIPK-1 and mixed lineage kinase domain like pseudokinase (MLKL), as well as any necrosome formation. Therefore, the enhanced cytotoxic effect of GEF under AAS culture conditions is thought to be mediated by atypical necroptosis.

Targeted Delivery of Auristatin-Modified Toxins to Pancreatic Cancer Using Aptamers.

Pancreatic cancer is one of the most lethal malignancies. Treatment with the first-line agent, gemcitabine, is often unsuccessful because it, like other traditional chemotherapeutic agents, is non-specific, resulting in off-target effects that necessitate administration of subcurative doses. Alternatively, monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF) are highly toxic small molecules that require ligand-targeted delivery. MMAE has already received FDA approval as a component of an anti-CD30 antibody-drug conjugate, brentuximab vedotin. However, in contrast to antibodies, aptamers have distinct advantages. They are chemicals, which allows them to be produced synthetically and facilitates the rapid development of diagnostics and therapeutics with clinical applicability. In addition, their small size allows for enhanced tissue distribution and rapid systemic clearance. Here, we assayed the toxicity of MMAE and MMAF conjugated to an anti-transferrin receptor aptamer, Waz, and an anti-epidermal growth factor receptor aptamer, E07, on the pancreatic cancer cell lines Panc-1, MIA PaCa-2, and BxPC3. In vitro, our results indicate that these aptamers are a viable option for the targeted delivery of toxic payloads to pancreatic cancer cells.

Abstract 633: Induction of ADCC resistance profoundly alters tumor cell phenotype and stress response

Abstract Antibody dependent cellular cytotoxicity (ADCC) is one mechanism by which monoclonal antibodies (mAb) work. In addition to ADCC, mAbs act on their target by causing signal perturbation and complement activation. Cetuximab is an EGFR targeting mAb used to target EGFR overexpression and amplification seen in many types of cancer. While mechanisms of resistance to EGFR targeted therapy have been extensively studied, resistance to ADCC has not, mainly due to the lack of ADCC-resistance models for study. To address this limitation we established a model system for anti-EGFR ADCC using NK92-CD16V effector cells, cetuximab, and the high EGFR-expressing squamous cell carcinoma cell line A431. Continuous A431 exposure to ADCC yielded an ADCC resistant phenotype (ADCCr) that exhibits a stable phenotype in the absence of continued ADCC selection. We have explored: ADCCr cell gene expression profile, NK cell activation, metabolic signature and subsequent RPPA analysis to further understand the causes and properties associated with this resistance. This ADCCr cell line has a distinctive transcriptional profile highlighted by overexpression of histone- and interferon-related genes, reduced sensitivity to antimetabolites, DNA-intercalating and ABC transporter-regulated cytotoxic agents. Intense ADCC selection causes epigenetic modification and stress response characterized by the transcriptional overexpression of PCAF (KAT2B), which initiates histone hyper-acetylation and epigenetic changes, inducing DNA replication arrest, DNA damage and stress responses that activate checkpoint signaling in the cell cycle. The pharmacologic inhibition of KAT2B reverses the ADCCr phenotype. Immune checkpoints such as PD-L1 do not modulate ADCC in this model system. These results shed light on new mechanisms of ADCC resistance and inform future combinatorial treatments for mAb therapy. We are exploring the possibility that stress response mechanisms are responsible for resistance for diverse selection pressures imposed by immune synapse-mediated cytotoxic attack. Citation Format: Dalal S. Aldeghaither, Joseph C. Murray, Sarah M. Roth, Elana J. Fertig, Shaojun Tang, Sandra A. Jablonski, Louis M. Weiner. Induction of ADCC resistance profoundly alters tumor cell phenotype and stress response [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 633. doi:10.1158/1538-7445.AM2017-633

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.

Induction of tumor cell-based ADCC resistance to create new tumor cell survival and therapy resistance mechanisms.

130 Background: EGFR overexpression &amp; amplification is seen in many types of cancer. Cetuximab is an EGFR targeting monoclonal antibody that acts by signal perturbation, complement activation &amp; inducing antibody dependent cellular cytotoxicity(ADCC) by recruiting effector cells. Cetuximab resistance has been extensively investigated, but it has proven difficult to study the molecular mechanisms underlying ADCC resistance because such models are not readily available. Methods: We established an EGFR-targeted ADCC resistance model system using NK92-CD16V effector cells, Cetuximab, and the A431 EGFR-expressing squamous cell carcinoma cell line. Continuous ADCC challenge of A431 (26+ passages) yielded an ADCC-resistant phenotype (ADCCr) that exhibits a stable phenotype in the absence of continued ADCC selection. To further understand the evolution of resistance to ADCC, we explored the ADCCr cell surface signature, gene expression profile &amp; ability to activate NK cells. Results: We find that ADCCr cells(when compared to its ADCC-sensitive(ADCCs) counterpart) have a modest reduction of EGFR expression, a distinctive transcriptional profile highlighted by overexpression of histone &amp; interferon related genes, altered cell surface expression of important cell adhesion molecules, reduced in vitro proliferation and in vivo growth &amp; resistance that is specific to the formation and function of immune synapses. ADCCr can be partially reversed by the exposure to an inhibitor of the histone acetyl transferase p300 subunit. Interestingly, the ADCCr cell line has reduced sensitivity (compared with ADCCs) to antimetabolite, DNA-intercalating and ABC transporter-regulated cytotoxic agents.The PD-L1 immune checkpoint does not modulate ADCC in this model system. Conclusions: These findings suggest that the induction of resistance to EGFR-targeted immune attack in this model system creates broadly useful survival and potential therapy resistance mechanisms.

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 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 (&amp;gt;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&amp;lt;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&amp;lt;0.0001), 2-fold reduction in mRNA level (qPCR, p&amp;lt; 0.01) and 1.8-fold reduction in protein level (immunoblot densitometry, p&amp;lt;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.

PET imaging of epidermal growth factor receptor expression in tumours using 89Zr-labelled ZEGFR:2377 affibody molecules.

Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor, which is overexpressed in many types of cancer. The use of EGFR-targeting monoclonal antibodies and tyrosine-kinase inhibitors improves significantly survival of patients with colorectal, non-small cell lung cancer and head and neck squamous cell carcinoma. Detection of EGFR overexpression provides important prognostic and predictive information influencing management of the patients. The use of radionuclide molecular imaging would enable non-invasive repeatable determination of EGFR expression in disseminated cancer. Moreover, positron emission tomography (PET) would provide superior sensitivity and quantitation accuracy in EGFR expression imaging. Affibody molecules are a new type of imaging probes, providing high contrast in molecular imaging. In the present study, an EGFR-binding affibody molecule (ZEGFR:2377) was site-specifically conjugated with a deferoxamine (DFO) chelator and labelled under mild conditions (room temperature and neutral pH) with a positron-emitting radionuclide 89Zr. The 89Zr-DFO-ZEGFR:2377 tracer demonstrated specific high affinity (160±60 pM) binding to EGFR-expressing A431 epidermoid carcinoma cell line. In mice bearing A431 xenografts, 89Zr-DFO-ZEGFR:2377 demonstrated specific uptake in tumours and EGFR-expressing tissues. The tracer provided tumour uptake of 2.6±0.5% ID/g and tumour-to-blood ratio of 3.7±0.6 at 24 h after injection. 89Zr-DFO-ZEGFR:2377 provides higher tumour-to-organ ratios than anti-EGFR antibody 89Zr-DFO-cetuximab at 48 h after injection. EGFR-expressing tumours were clearly visualized by microPET using 89Zr-DFO-ZEGFR:2377 at both 3 and 24 h after injection. In conclusion, 89Zr-DFO-ZEGFR:2377 is a potential probe for PET imaging of EGFR-expression in vivo.

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