EGFR-targeted Antibodies Research Articles

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.

Correction to: A phase I study of LY3164530, a bispecific antibody targeting MET and EGFR, in patients with advanced or metastatic cancer.

The original version of this article unfortunately contained a mistake. The co-authors' names were incorrect.

Epidermal Growth Factor Receptor Extracellular Domain Mutations in Glioblastoma Present Opportunities for Clinical Imaging and Therapeutic Development

We explored the clinical and pathological impact of epidermal growth factor receptor (EGFR) extracellular domain missense mutations. Retrospective assessment of 260 de novo glioblastoma patients revealed a significant reduction in overall survival of patients having tumors with EGFR mutations at alanine 289 (EGFRA289D/T/V). Quantitative multi-parametric magnetic resonance imaging analyses indicated increased tumor invasion for EGFRA289D/T/V mutants, corroborated in mice bearing intracranial tumors expressing EGFRA289V and dependent on ERK-mediated expression of matrix metalloproteinase-1. EGFRA289V tumor growth was attenuated with an antibody against a cryptic epitope, based on in silico simulation. The findings of this study indicate a highly invasive phenotype associated with the EGFRA289V mutation in glioblastoma, postulating EGFRA289V as a molecular marker for responsiveness to therapy with EGFR-targeting antibodies.

Abstract 2786: Pharmacokinetics and in vitro/in vivo characterization of high-affinity bispecific EGFR/CD16A NK cell engagers for the treatment of EGFR-expressing tumors

Abstract The epidermal growth factor receptor (EGFR) is a validated target for the treatment of several solid tumor types. Current EGFR-targeting monoclonal antibodies (mAbs) and tyrosine kinase inhibitors function mainly through blocking of signal-transduction. Moreover, treatment with these agents is dependent on the receptor's mutational status which may cause treatment resistance in a large number of patients. In addition, EGFR-targeting therapies have been associated with side effects considered to impact prescription rates, in particular in the U.S. A natural killer (NK) cell-based EGFR-targeting approach has the potential to widen the therapeutic window and overcome intrinsic and acquired resistance. Therefore, it offers a promising and well differentiated therapeutic option. We describe the engineering of different bispecific NK cell engagers designed to redirect NK cell-mediated killing to EGFR-positive tumors. Different bispecific EGFR/CD16A antibody constructs targeting human and cynomolgus EGFR and CD16A were designed using novel human Fv antibody binding domains from a toolbox of formats. The Fv domains were evaluated in terms of their pharmacokinetic (PK) properties. Furthermore, the antibody constructs were characterized in antigen and cell binding assays, as well as in in vitro cytotoxicity assays against tumor cell lines expressing Raswt or mutated Ras. PK parameters were determined in CD1 mice and in vivo efficacy of selected antibodies was investigated in an A-431 tumor model in humanized mice. We generated antibody constructs with varying half-lives, including antibodies with a PK profile which allows for dosing comparable to IgG-based antibodies, while aiming at an improved safety profile compared to other EGFR-targeting therapies. Different high affinity tetravalent, bispecific antibodies binding to CD16A and the extracellular domain of EGFR were characterized. While binding to CD16A of classical mAbs is impaired by serum IgG, no substantial changes in binding affinity of our constructs to NK cells were observed. The antibodies showed superior potency and efficacy compared to mAbs or Fc-enhanced antibodies when tested in cytotoxicity assays. In vivo efficacy of selected antibodies was demonstrated in an A-431 tumor model in humanized mice. We have successfully designed and characterized several novel, highly potent and differentiated tetravalent bispecific antibody constructs to redirect NK cell-mediated cell killing to EGFR-positive tumor cells. These drug candidates are suitable for the treatment of EGFR-expressing malignancies with the potential to overcome resistance to other EGFR-targeting agents and offering a potentially improved safety profile. These candidates are currently in development as monotherapy and are also explored in combination with immune activating agents. Citation Format: Michael Kluge, Michael Tesar, Uwe Reusch, Stefan Knackmuss, Torsten Haneke, Kristina Ellwanger, Ivica Fucek, Thomas Mueller, Ute Schniegler-Mattox, Martin Treder. Pharmacokinetics and in vitro/in vivo characterization of high-affinity bispecific EGFR/CD16A NK cell engagers for the treatment of EGFR-expressing tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2786.

Abstract 2600: Resistance biomarkers in patients with head and neck squamous cell carcinoma treated with cetuximab

Abstract BACKGROUND: Cetuximab, a monoclonal antibody targeting EGFR, improves overall survival in the locally advanced setting in combination with radiotherapy and in first-line recurrent setting in combination with chemotherapy in HNSCC patients. No predictive biomarker of efficacy of cetuximab in these settings. We aimed at identifying biomarkers of sensitivity/resistance to cetuximab. PATIENTS AND METHODS: HNSCC patients treated with cetuximab at Institut Curie, for which complete clinicopathological data and formalin-fixed, paraffin-embedded (FFPE) tumor tissue were available and collected before cetuximab treatment, were included in this retrospective study. Immunohistochemistry analyses of PTEN and EGFR were performed to assess expression levels. PIK3CA and KRAS mutations were analyzed using high resolution melting (HRM) and Sanger sequencing. The predictive value of these alterations on progression-free survival (PFS) was evaluated. RESULTS: PIK3CA and KRAS/HRAS mutations were associated with poor PFS in HNSCC patients treated with cetuximab in first-line recurrence setting but not in patients treated with cetuximab in combination with radiotherapy. PTEN loss of expression had a negative prognostic impact in HNSCC patients treated with cetuximab and radiotherapy, while EGFR high expression was not found to be a sensitivity biomarker for cetuximab in our patient population. CONCLUSIONS: PIK3CA and RAS mutations were resistance biomarkers of cetuximab in first-line recurrent setting, whereas PTEN loss was a resistance biomarker of cetuximab when given in combination with radiotherapy. Citation Format: Maud Kamal, Olivia Leblanc, Sophie Vacher, Charlotte Lecerf, Emmanuelle Jeannot, Jerzy Klijanienko, Frederique Berger, Caroline Hoffmann, Valentin Calugaru, Nathalie Badois, Anne Chilles, Maria Lesnik, Samar Krhili, Thomas Jouffroy, Ivan Bieche, Christophe Le Tourneau. Resistance biomarkers in patients with head and neck squamous cell carcinoma treated with cetuximab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2600.

Abstract LB-340: Combinatorial platform for CART cell therapy for glioblastoma

Abstract Introduction: Our clinical program in chimeric antigen receptor T (CART) cell therapy has focused on targeting EGFR oncoproteins for glioblastoma (GBM). We have completed a phase I trial using CART cells directed to the EGFRvIII antigen and showed in situ uptake, proliferation, functional activation and antigen editing by CART cells in GBM tissue. This first clinical trial demonstrated two barriers to clinical efficacy. First, GBM heterogeneity was striking spatially and temporally, including the number of variants in EGFR. Second, CART cell therapy was associated with an adaptive anti-GBM response illustrated by an initial wave of CART activation followed by dramatic immunosuppression. These two features indicate that we need strategies to overcome GBM heterogeneity and the immunosuppressive tumor microenvironment (TME). Methods: We have used multiple CART variants, targeting both IL13Rα2 and EGFRvIII, in combination with selected immune checkpoint blockade inhibitors, to explore possible additive effects in both in vitro and in vivo GBM model systems on limiting the immunosuppressive GBM TME. Independently, we have developed a portfolio of CARTs with different binding specificities that target other homo- and heterodimers of EGFR oncoproteins, to address GBM heterogeneity. Results: Combination studies of CART cells with immune checkpoint blockade inhibitors revealed a non-homogenous response. Different CART structures and targets showed increased tumor killing activity with specific immune checkpoint blockade inhibition. Specifically, IL13Rα2-targeting CART cells had the best effect when paired with anti-CTLA4 treatment, while EGFRvIII-targeting CART cells had the largest effect when paired with anti-PD-1 treatment. In parallel to our combination therapy work, we have expanded our repertoire of CART constructs for targeting EGFR oncogenic alleles with extracellular domain mutations by utilizing antibody phage display technology. EGFR-targeting antibody sequences were incorporated into viral constructs and transduced into T cells. We have shown variable killing with these novel CART constructs in GSC/PDX models of GBM heterogeneity. Conclusion: Our initial experience with CART cells in GBM suggested that although a single intravenous infusion results in CART cell bioactivity in the brain, overcoming the adaptive changes in the local TME and addressing antigen heterogeneity may improve the clinical efficacy of CART-directed strategies. Our combination work showed that different CART constructs cooperate with immune checkpoint blockade inhibition differentially. This selective pairing suggests that a more personalized immunophenotypic assessment may result in higher efficacy therapeutic combinations. In addition, development of a broad portfolio of both selective and promiscuous CART constructs targeting EGFR in its various forms gives us the potential to cover a larger percentage of both the GBM tumor volume and regionally-specific tumor cells within a single GBM. Citation Format: Zev A. Binder, Yibo Yin, Radhika Thokala, Donald M. O'Rourke. Combinatorial platform for CART cell therapy for glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-340.

Characterization of ABBV-221, a Tumor-Selective EGFR-Targeting Antibody Drug Conjugate.

Depatuxizumab mafodotin (depatux-m, ABT-414) is a tumor-selective antibody drug conjugate (ADC) comprised of the anti-EGFR antibody ABT-806 and the monomethyl auristatin F (MMAF) warhead. Depatux-m has demonstrated promising clinical activity in glioblastoma multiforme (GBM) patients and is currently being evaluated in clinical trials in first-line and recurrent GBM disease settings. Depatux-m responses have been restricted to patients with amplified EGFR, highlighting the need for therapies with activity against tumors with nonamplified EGFR overexpression. In addition, depatux-m dosing has been limited by corneal side effects common to MMAF conjugates. We hypothesized that a monomethyl auristatin E (MMAE) ADC utilizing an EGFR-targeting antibody with increased affinity may have broader utility against tumors with more modest EGFR overexpression while mitigating the risk of corneal side effects. We describe here preclinical characterization of ABBV-221, an EGFR-targeting ADC comprised of an affinity-matured ABT-806 conjugated to MMAE. ABBV-221 binds to a similar EGFR epitope as depatux-m and retains tumor selectivity with increased binding to EGFR-positive tumor cells and greater in vitro potency. ABBV-221 displays increased tumor uptake and antitumor activity against wild-type EGFR-positive xenografts with a greatly reduced incidence of corneal side effects relative to depatux-m. ABBV-221 has similar activity as depatux-m against an EGFR-amplified GBM patient derived xenograft (PDX) model and is highly effective alone and in combination with standard-of-care temozolomide in an EGFRvIII-positive GBM xenograft model. Based on these results, ABBV-221 has advanced to a phase I clinical trial in patients with advanced solid tumors associated with elevated levels of EGFR. Mol Cancer Ther; 17(4); 795-805. ©2018 AACR.

A novel bispecific antibody fusion protein co-targeting EGFR and CD47 with enhanced therapeutic index.

To promote targeting specificity of anti-CD47 agents, we have constructed a novel bispecific antibody fusion protein against EGFR and CD47, which may minimize the "off-target" effects caused by CD47 expression on red blood cells. The novel bispecific antibody fusion protein, denoted as Bi-SP could simultaneously bind to EGFR and CD47 and exhibited potent phagocytosis-stimulation effects in vitro. Bi-SP treatment with a low dose more effectively inhibited tumor growth than either EGFR-targeting antibody, Pan or the SIRPα variant-Fc (SIRPαV-Fc) in the A431 xenograft tumor model. In addition, the treatment with Bi-SP produced less red blood cell (RBC) losses than the SIRPαV-Fc treatment, suggesting its potential use for minimizing RBC toxicity in therapy. Bi-SP with improved therapeutic index has the potential to treat CD47+ and EGFR+ cancers in clinics.

Characterization of 7A7, an anti-mouse EGFR monoclonal antibody proposed to be the mouse equivalent of cetuximab.

The Epidermal Growth Factor Receptor (EGFR) is selectively expressed on the surface of numerous tumours, such as non-small cell lung, ovarian, colorectal and head and neck carcinomas. EGFR has therefore become a target for cancer therapy. Cetuximab is a chimeric human/mouse monoclonal antibody (mAb) that binds to EGFR, where it both inhibits signaling and induces cell death by antibody-dependent cell mediated cytotoxicity (ADCC). Cetuximab has been approved for clinical use in patients with head and neck squamous cell carcinoma (HNSCC) and colorectal cancer. However, only 15-20% patients benefit from this drug, thus new strategies to improve cetuximab efficiency are required. We aimed to develop a reliable and easy preclinical mouse model to evaluate the efficacy of EGFR-targeted antibodies and examine the immune mechanisms involved in tumour regression. We selected an anti-mouse EGFR mAb, 7A7, which has been reported to be “mouse cetuximab” and to exhibit similar properties to its human counterpart. Unfortunately, we were unable to reproduce previous results obtained with the 7A7 mAb. In our hands, 7A7 failed to recognize mouse EGFR, both in native and reducing conditions. Moreover, in vivo administration of 7A7 in an EGFR-expressing HPV38 tumour model did not have any impact on tumour regression or animal survival. We conclude that 7A7 does not recognize mouse EGFR and therefore cannot be used as the mouse equivalent of cetuximab use in humans. As a number of groups have spent effort and resources with similar issues we feel that publication is a responsible approach.

Abstract A089: Exploiting clonal evolution and liquid biopsy to overcome resistance to anti-EGFR treatment in metastatic colorectal cancer: the CHRONOS trial

Abstract Background: The EGFR-targeted antibodies cetuximab and panitumumab (MAB-EGFRs) are used for the treatment of RAS-axis wild-type metastatic colorectal cancer (mCRC). Unfortunately, acquired resistance to MAB-EGFRs inevitably develops, dramatically curtailing their clinical use (Misale et al., Cancer Discov 2014). We found that mutations in KRAS, NRAS, and BRAF are the main culprit of this acquired resistance, and that high ctDNA levels of these mutated genes can be found in plasma of progressing patients. However, in preclinical models made resistant to cetuximab, acquired RAS-axis mutated clones decay upon cetuximab withdrawal, allowing tumor cells to regain sensitivity to further anti-EGFR treatment. Paralleling this behavior, RAS-axis WT patients who occasionally benefited from a rechallenge treatment with MAB-EGFRs exhibited corresponding pulsatile levels of mutant RAS ctDNA (Siravegna et al., Nat Med 2015). Collectively, these results suggest that the CRC genome adapts dynamically to intermittent MAB-EGFRs schedules, and provide a molecular rational for rechallenge therapies based on EGFR blockade. Methodology: CHRONOS is a liquid biopsy-driven trial to assess the efficacy of rechallenging with panitumumab (PAN) mCRC patients with ct-DNA-proven RAS-mediated acquired resistance. CHRONOS patients’ selection is based on a progressive enrichment strategy. In the SCREENING phase, multiple wild-type patients [MWTP, i.e., patients not harboring any drivers of resistance to MAB-EGFRs at tumor level (Bertotti et al., Nature 2015)] are identified upfront with a multiplex mCRC-specific tissue-genotyping panel (FUNNEL). In the MOLECULAR phase, MWTP responding to a 1st-line treatment inclusive of MAB-EGFR and developing RAS-mutated ctDNA clones at progression (PD), start 2nd-line chemotherapy and are blood monitored for RAS-related clones' decay. MWTP experiencing a &amp;gt;50% drop in ctDNA RAS levels at 2nd-line PD enter the TRIAL phase and are rechallenged with PAN. The main endpoint is objective response (OR), aiming to achieve a 30% OR rate. 129 MWTP responding and progressing to 1st-line MAB-EGFR-based therapy will be blood-monitored from first to second PD. We expect to find detectable (&amp;gt;3 MAF) ctDNA RAS mutated clones in 80% of cases (N 36), decaying &amp;gt;50% during 2nd-line "EGFR-therapy holiday" (60% of cases). These “ctDNA eligible” patients will receive PAN at 6 mg/kg Q2 weeks until PD or unacceptable toxicity. 27 MWTP will be recruited in TRIAL phase with 6 responses necessary to declare the study positive (β 0.15; α 0.05). Liquid biopsy will be used to correlate the decay kinetics of RAS-extended clone(s) during MAB-EGFR treatment holiday with response to PAN rechallenge, and to link the blood presence of RAS-extended clone(s) during PAN treatment with response and its duration. The tumor ctDNA plasma will be analysed by NGS before and after PAN rechallenge and potential associations identified between different mutated ctDNA clones (RAS or others) and response to PAN rechallenge. Conclusion: This is the 1st trial in mCRC exploiting liquid biopsy and the clonal evolution of tumor cell under treatment pressure, to extend the use of anti-EGFR therapy beyond the point of clinically established resistance in mCRC. (NCT03227926.) Funded by AIRC grant #9970. Citation Format: Salvatore Siena, Alberto Bardelli, Andrea Sartore-Bianchi, Sara Lonardi, Francesco Leone, Francesca Bergamo, Giuseppe Tonini, Filippo De Braud, Filippo Pietrantonio, Lorenza Rimassa, Armando Santoro, Giulia Siravegna, Benedetta Mussolin, Francesco Rua, Erica Francesca Bonazzina, Alessio Amatu, Patrizia Racca, Andrea Ardizzoni, Vittorina Zagonel, Silvia Marsoni. Exploiting clonal evolution and liquid biopsy to overcome resistance to anti-EGFR treatment in metastatic colorectal cancer: the CHRONOS trial [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A089.

Evaluation of Early Therapeutic Effects after Near-Infrared Photoimmunotherapy (NIR-PIT) Using Luciferase-Luciferin Photon-Counting and Fluorescence Imaging.

Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that induces highly selective immunogenic cell death. It is based on an antibody-photoabsorber conjugate (APC) that is activated by NIR light. The purpose of this study was to investigate the effects of NIR-PIT as measured by luciferase-luciferin photon-counting and fluorescence imaging. Six days after subcutaneous injection of A431-luc-GFP cells tumors formed in a xenograft mouse model. The EGFR-targeting antibody, panitumumab, was conjugated to the photoabsorber, IRDye-700DX (pan-IR700), and was intravenously administered to tumor-bearing mice. Serial luciferase-luciferin photon-counting images and both green fluorescent protein (GFP) and IR700 fluorescence images were obtained from the same mice before and after NIR-PIT treatment (0, 10, 20, 30 min (early phase), and 24, 48 h (late phase) after NIR light exposure). Optical signal intensities were compared for each modality. IR700 fluorescence and luciferase-luciferin photon-counting images showed decreased intensities in both the early and late phases after NIR-PIT (p < 0.01). On the other hand, GFP fluorescence images showed decreased intensities only in the late phase (p < 0.01). In the early phase, GFP fluorescence images showed smaller intensity reductions compared to IR700 fluorescence and luciferase-luciferin photon-counting (p < 0.01), while in the late phase, IR700 fluorescence showed smaller intensity reductions than luciferase-luciferin photon-counting and GFP fluorescence (p < 0.05), due to redistribution of pan-IR700 within the tumor bed. In conclusion, luciferase-luciferin photon-counting imaging is suitable to evaluate early phase NIR-PIT effects, while both luciferase-luciferin photon-counting and GFP reflected later phase effects.

EGFR Gene Copy Number by FISH May Predict Outcome of Necitumumab in Squamous Lung Carcinomas: Analysis from the SQUIRE Study.

Necitumumab is a monoclonal antibody targeting EGFR. In the SQUIRE trial, the addition of necitumumab to chemotherapy for squamous cell lung cancer significantly improved overall survival (OS) (hazard ratio [HR]= 0.84); in a post hoc analysis, EGFR copy number gain determined by fluorescence in situ hybridization (FISH) showed a trend toward improved OS (HR= 0.70) and progression-free survival (PFS) (HR= 0.71) with the addition of necitumumab. We present the analysis of granular EGFR FISH data from SQUIRE to examine the potential predictive role of high polysomy and gene amplification, as both were included in the FISH-positive category. Available specimens from SQUIRE underwent FISH analysis in a central laboratory, and each sample was evaluated by using the Colorado EGFR scoring criteria. Thecorrelation of granular FISH parameters with clinical outcomes was assessed. Samples were available for 557 of 1093 patients; 208 patients (37.3%) were FISH-positive, including 167 (30.0%) with high polysomy and 41 (7.4%) with gene amplification. In patients with high polysomy, the addition of necitumumab resulted in a statistically significant increase in PFS (6.08 versus 5.13 months [p= 0.044]) and nonstatistically significant increase in OS (12.6 versus 9.5 months [p= 0.133]); among patients with gene amplification, the addition of necitumumab did not significantly improve PFS (7.4 versus 5.6 months; [p= 0.334]) but did improve OS (14.8 versus 7.6 months; [p = 0.033]). EGFR copy number gain by FISH might have arole as a predictive biomarker for necitumumab in squamous cell lung cancer. In our opinion, these data encourage further studies to prospectively evaluate this potential biomarker.

Abstract 32: Preclinical evaluation of MCLA-158: A bispecific antibody targeting LGR5 and EGFR using patient-derived colon carcinoma organoids

Abstract Background. Colorectal cancer (CRC) is the third most common cancer and remains a large unmet need. Dysregulation of Wnt and receptor tyrosine kinase (RTK) signalling pathways are important oncogenic driving events in CRC. Due to this dysregulation, Wnt target genes are expressed at higher levels in CRC particularly in tumor initiating cells. We previously performed an unbiased screen of bispecific antibodies (bAbs) targeting Wnt and RTK targets that resulted in the selection of MCLA-158. Methods. A cohort of 32 genetically and transcriptionally annotated patient-derived colorectal cancer and normal colon organoids were used to functionally characterize responses to antibodies based on morphological changes with high content 3D imaging. Binding affinity was measured by surface plasma resonance and cell based assays. The antibody binding epitopes were mapped by shotgun mutagenesis and FACS based screening. Ligand (R-spondin or EGF) blocking activity was measured in vitro by competition for ligand binding or functional inhibition of ligand dependent growth. In vivo activity was evaluated in xenograft models generated from organoids subcutaneously implanted into immunocompromised mice. Safety was evaluated via once weekly intravenous administration of MCLA-158 to cynomolgus monkeys for 4 weeks and monitoring for pathological changes. Results. MCLA-158, an ADCC enhanced common light chain IgG1 bispecific antibody, binds in domain III of EGFR and in the N-Cap/1st LRR of LGR5, both ligand binding regions, however, only EGF binding was blocked by MCLA-158. MCLA-158 demonstrated inhibitory activity in 74% of tumor organoids independent of KRAS mutational status but was not active on organoids of the cohort harboring both KRAS and PIK3CA mutations. MCLA-158 was significantly more active on organoids derived from tumors than from normal tissue in contrast to cetuximab, which demonstrated equivalent activity on both (range 20-100 fold, n=4). In vivo activity was evaluated against tumor organoids with different KRAS mutation status shown to be sensitive to MCLA-158 in vitro. In all cases, MCLA-158 significantly inhibited the growth of the tumor compared to both control and cetuximab treatment. Inhibitors of both the Wnt and EGFR pathways have shown significant toxicity in humans. An initial evaluation of MCLA-158 toxicity in cynomolgus monkeys did not demonstrate any pathological finding after repeated dosing at 25mg/kg. Conclusions. MCLA-158 demonstrates superior activity compared to reference antibodies in both in vitro and in vivo tumor organoid based assays regardless of KRAS status and was well tolerated in non-human primates. These preclinical data suggest MCLA-158 could benefit patients with metastatic CRC and warrant clinical evaluation. Citation Format: Rob Roovers, Bram Herpers, Mark James, Berina Eppink, Carme Cortina, David Maussang-Detaille, Ingrid Kolfschoten, Sylvia Boy, Marc van de Wetering, Wim De Lau, Robert Doornbos, Kuan Yan, Lucia Salinaro, Lex Bakker, john de Kruif, Hans Clevers, Robert Vries, Eduard Batlle, Leo Price, Mark Throsby. Preclinical evaluation of MCLA-158: A bispecific antibody targeting LGR5 and EGFR using patient-derived colon carcinoma organoids [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 32. doi:10.1158/1538-7445.AM2017-32

Abstract 3697: The impact of HER3 signaling mediated PD-L1 regulation in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is still difficult to treat partly because of lacking specific target. Although 50-70% of TNBC expresses EGFR, it is less sensitive to the treatment of EGFR inhibition for TNBC as compared to the efficacy of HER2 inhibition for HER2-positive breast cancer. Several phase II study on EGFR blockade treatment has been reported, however it has not applied in a clinical setting yet. It was reported that residual tumors after treatment with EGFR-targeted antibodies showed increased HER3 abundance leading to EGFR/HER3 receptor dimerization. The signals of HER3/EGFR dimerization to PI3K/AKT/mTOR pathways are thought to be involved in cancer survival, proliferation and also up-regulation of PD-L1 expression. Thus, we hypothesized that up-regulation of HER3 signal caused by anti-cancer treatment might induce PD-L1 expression and inhibit host anti-tumor immunity. In this study, we tested the relationships between HER3 signal and PD-L1 expression by using three basal-like breast cancer cell line; MDA-MB-231, HCC70, and MDA-MB-468. MDA-MB-231 is HER3-negative, and HCC70 and MDA-MB-468 are HER3-positive cell lines. We added neureglin 1 (NRG1: HER3 ligand) to those three cell lines and analyzed PD-L1 expression of protein by flowcytometry and mRNA by qRT-PCR. Both protein and mRNA level of PD-L1 on HCC70 and MDA-MB-468 treated with NRG1 are increased as compared with those without NRG1 while there was no change of PD-L1 expression of MDA-MB-231 either with or without NRG1. In order to confirm the significance of potential treatment target of HER3, we evaluated HER3 expression in biopsy samples by immunohistochemistry before neoadjuvant chemotherapy (NAC) including all phenotypes. Thirteen pathological complete response (pCR) cases after NAC and 6 non pCR cases were included. We scored the HER3 stainability from 0 to 3 and found that non pCR cases showed significantly higher HER3 score than pCR cases (84.6% and 33% respectively, p=0.0149). Although further study is needed, these results suggest that HER3 signal possibly regulates PD-L1 expression in HER3-positive basal-like breast cancer and treatment with anti-HER3 targeting therapy combination with an immune checkpoint inhibition therapy for HER3 positive NAC resistant patients might be warranted. Note: This abstract was not presented at the meeting. Citation Format: Ayane Yamaguchi, Eiji Suzuki, Kosuke Kawaguchi, Mariko Nishie, Moe Tsuda, Takeshi Kotake, Masakazu Toi. The impact of HER3 signaling mediated PD-L1 regulation in triple negative breast cancer [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 3697. doi:10.1158/1538-7445.AM2017-3697

TMEM16A/ANO1 suppression improves response to antibody-mediated targeted therapy of EGFR and HER2/ERBB2.

TMEM16A, a Ca2+ -activated Cl- channel, contributes to tumor growth in breast cancer and head and neck squamous cell carcinoma (HNSCC). Here, we investigated whether TMEM16A influences the response to EGFR/HER family-targeting biological therapies. Inhibition of TMEM16A Cl- channel activity in breast cancer cells with HER2 amplification induced a loss of viability. Cells resistant to trastuzumab, a monoclonal antibody targeting HER2, showed an increase in TMEM16A expression and heightened sensitivity to Cl- channel inhibition. Treatment of HNSCC cells with cetuximab, a monoclonal antibody targeting EGFR, and simultaneous TMEM16A suppression led to a pronounced loss of viability. Biochemical analyses of cells subjected to TMEM16A inhibitors or expressing chloride-deficient forms of TMEM16A provide further evidence that TMEM16A channel function may play a role in regulating EGFR/HER2 signaling. These data demonstrate that TMEM16A regulates EGFR and HER2 in growth and survival pathways. Furthermore, in the absence of TMEM16A cotargeting, tumor cells may acquire resistance to EGFR/HER inhibitors. Finally, targeting TMEM16A improves response to biological therapies targeting EGFR/HER family members.

Near Infrared Photoimmunotherapy in a Transgenic Mouse Model of Spontaneous Epidermal Growth Factor Receptor (EGFR)-expressing Lung Cancer.

Near infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment that combines the specificity of antibodies for targeting tumors with the toxicity induced by a sensitive photoabsorber following exposure to NIR light. Most studies of NIR-PIT have been performed in xenograft models of cancer. The purpose of this study was to evaluate the therapeutic effects of NIR-PIT in a transgenic model of spontaneous lung cancer expressing human EGFR (hEGFR-TL). Mice were separated into 3 groups for the following treatments: (1) no treatment (control); (2) 150 μg of photoabsorber, IR700, conjugated to panitumumab, an antibody targeting EGFR [antibody-photoabsorber conjugate (APC)] intravenously (i.v.) only; (3) 150 μg of APC i.v. with NIR light administration. Each treatment was performed every week up to three weeks. MRI was performed 1 day before and 3, 6, 13, 20, 27, and 34 days after first NIR-PIT. The relative volume of lung tumors was calculated from the tumor volume at each MRI time point divided by the initial volume. Steel test for multiple comparisons was used to compare the tumor volume ratio with that of control. Tumor volume ratio was inhibited significantly in the NIR-PIT group compared with control group (P < 0.01 at all time points). In conclusion, NIR-PIT effectively treated a spontaneous lung cancer in a hEGFR-TL transgenic mouse model. MRI successfully monitored the therapeutic effects of NIR-PIT. Mol Cancer Ther; 16(2); 408-14. ©2016 AACR.

Influence of the HER receptor ligand system on sensitivity to cetuximab and trastuzumab in gastric cancer cell lines

PurposeGastric cancer remains a major health concern, and improvement of the therapeutic options is crucial. Treatment with targeted therapeutics such as the EGFR-targeting antibody cetuximab or the HER2-targeting antibody trastuzumab is either ineffective or moderately effective in this disease, respectively. In this study, we analysed the involvement of the HER receptor ligands amphiregulin (AREG), epidermal growth factor (EGF), heparin-binding epidermal growth factor (HB-EGF) and transforming growth factor alpha (TGFα) in the responsiveness of gastric cancer cell lines to cetuximab and trastuzumab.MethodsA panel of 11 gastric cancer cell lines was characterized for cetuximab and trastuzumab sensitivity, ligand secretion and expression and activation of the HER receptors using WST-1 cell proliferation assays, ELISAs and Western blot analyses. We further investigated the effects of an exogenous ligand application on the cetuximab and trastuzumab sensitivity.ResultsWe found no correlation between TGFα secretion and the sensitivity to cetuximab or trastuzumab. For AREG, we confirmed previous results indicating that this ligand is a positive predictor of cetuximab sensitivity. Exogenous HB-EGF was effective in rescuing sensitive cell lines from inhibition of cell proliferation by both, cetuximab and trastuzumab.ConclusionsOur data indicate that HB-EGF may be a useful marker for the prediction of trastuzumab sensitivity in gastric cancer.

Abstract 580: Anti-EGFRvIII TandAbs recruiting either T or NK cells are highly specific and potent therapeutic antibody candidates for the treatment of EGFRvIII+ tumors

Abstract EGFRvIII is the most prevalent tumor-specific variant of the wild-type EGFR and represents an attractive tumor target in various solid tumors such as GMB, HNSCC or NSCLC. Despite the clinical successes achieved with EGFR targeting antibodies or small molecule inhibitors little therapeutic progress has been made with EGFRvIII. There is still a high medical need in such cancers and several agents are in development to address this notoriously difficult target: Celldex′ vaccine rindopepimut in combination with bevacizumab in late-stage development, or ADCs in early development by Amgen or Abbvie. However, despite the high tumor specificity of EGFRvIII expression, no potent T- or NK-cell engaging are currently in clinical development. It has been recognized that there may be more therapeutic potential with bi-specific antibodies recruiting immune effectors for targeted destruction of antigen-positive tumor cells. We developed tetravalent, bi-specific TandAbs that recognize EGFRvIII, and recruiting either T-cells or NK-cells by binding to the activating receptors CD3 and CD16A, respectively. This targeted antibody approach allows the selective destruction of EGFRvIII+ tumor cells employing highly potent and efficacious immune effector cells whilst sparing normal cells that are EGFRvIII- or cells presenting the ubiquitously expressed EGFR. The selected EGFRvIII/CD3 and EGFRvIII/CD16A TandAbs exhibited exquisite specificity towards EGFRvIII in Western Blot, SPR, ELISA, and flow cytometric assays. No binding was observed to recombinant EGFR or to EGFR+ cells. They also displayed potent cytotoxicity towards EGFRvIII+ cell lines with EC50 values in the low picomolar range. No cytotoxicity was observed on EGFRvIII- target cells or cells expressing EGFR demonstrating the high selectivity of anti-EGFRvIII TandAbs. Importantly, in the absence of EGFRvIII+ target cells our TandAbs did not elicit T- or NK-cell activation and activation-induced immune cell proliferation suggesting an excellent safety profile. In vivo pharmacodynamics for anti-EGFRvIII TandAbs was demonstrated in a mouse xenograft model. The clinical relevance of EGFRvIII as a tumor marker and the binding of our anti-EGFRvIII variable domains were evaluated by immunohistochemistry. Binding was shown in glioblastoma and other solid tumors, but no expression was detectable on healthy tissue. In summary, the presented in vitro and in vivo studies qualify EGFRvIII/CD3 and EGFRvIII/CD16A TandAbs as highly attractive therapeutic antibody candidates and provides us with the possibility of employing both T-cell and NK-cell recruiting strategies for selective immunotherapy of EGFRvIII+ tumors. Due to tumor specific expression of EGFRvIII and the absence of off-target activity our TandAbs display an excellent safety profile reducing the risks of side effects associated with other anti-EGFRvIII therapies. Citation Format: Kristina Ellwanger, Uwe Reusch, Ivica Fucek, Michael Weichel, Thorsten Gantke, Stefan Knackmuss, Martin Treder. Anti-EGFRvIII TandAbs recruiting either T or NK cells are highly specific and potent therapeutic antibody candidates for the treatment of EGFRvIII+ tumors. [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 580.

Abstract 3502: Combination of BKM120 and erlotinib in squamous cell carcinoma of the head and neck: mechanism of in vitro and in vivo synergy

Abstract Purpose: Molecularly targeted agents will play a major role in the next generation of personalized cancer therapies. The EGFR-targeted monoclonal antibody cetuximab is currently the only FDA-approved targeted treatment for head and neck cancers (HNC) with a response rate of less than 15%. Recent large scale genomic studies including TCGA suggest that &amp;gt;50% of HNC cases have activation of the PI3K/AKT/mTOR pathways, suggesting PI3K as an excellent target for HNC. The purpose of the current study is to investigate whether co-targeting EGFR and PI3K has synergistic antitumor effects and to understand the mechanism of synergy. Methods: In a panel of 10 malignant and 1 premalignant HN cell lines, we evaluated cell growth inhibition (by SRB assay); IC50, combination index, and dose reduction index (by CalcuSyn), and apoptosis (by Annexin-V staining). mRNA and protein expression were measured by qPCR and Western blotting, respectively. Small molecule chemical inhibitors and siRNA-mediated knockdown strategies were used to inactivate and shut down the expression of the relevant proteins, respectively. Results: Single targeting of EGFR with erlotinib or PI3K with BKM120 (pan-PI3K inhibitor) suppressed cellular growth without inducing significant apoptosis. Co-targeting EGFR and PI3K had in vitro synergy in all except one cell line (based on combination index and dose reduction index) and more effectively inhibited HNC xenograft growth in vivo. The combination of erlotinib and BKM120 induced variable apoptosis: some cell lines were very sensitive (Tu686, 686LN, 93-VU-147T), some moderately (Fadu, SqCCy1, 1483, UMSSC90) and others were resistant (UD-SCC2, MSK-LEUK1, JHU022) to apoptosis induction. Only pan-PI3K inhibitors (BKM120 and BEZ235) induced effective apoptosis in combination with erlotinib, since the PI3K-α inhibitor BYL279 or allosteric mTORC1 inhibitor RAD001 failed to induce apoptosis in combination with erlotinib. Erlotinib strongly inhibited p-EGFR and p-ERK in vitro and in vivo, but only partially inhibited p-AKT. On the other hand, BKM120 completely inhibited p-AKT without affecting p-ERK. We also found that the combination of BKM120 and erlotinib strongly inhibited both axes of the AKT-mTORC1 pathway in sensitive cell lines, but failed to inhibit p-4EBP1 in one resistant cell line (UD-SCC2). In addition, the combination of BKM120 and erlotinib strongly inhibited Bcl-2, Bcl-xL and MCL-1 at the translational level in the sensitive cell lines but not the resistant one. siRNA-mediated knockdown of eIF4E (to inhibit 4EBP1-eIF4E-dependent translation) sensitized UD-SCC2 cells to the combination of erlotinib and BKM-induced apoptosis. Conclusions: Our results strongly suggest that co-targeting of EGFR and PI3K is synergistic and induces apoptosis of HNC cell lines by inhibiting both axes of the AKT-mTOR pathway and translational regulation of anti-apoptotic Bcl-2 proteins. Citation Format: Abu Anisuzzaman, Abedul Haque, Zhuo Chen, Dong M. Shin, A.R.M. Ruhul Amin. Combination of BKM120 and erlotinib in squamous cell carcinoma of the head and neck: mechanism of in vitro and in vivo synergy. [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 3502.

Abstract 1866: HER3 targeting sensitizes HNSCC to cetuximab - evidence from cell line and patient derived xenograft (PDX) models

Abstract Background and Objectives: Our previous study demonstrated that a combination of EGFR-targeted antibody cetuximab and HER3-specific antibody MM121 could significantly inhibit growth of head and neck squamous cell carcinoma (HNSCC) as compared with either of the single drugs. The current study aims to understand the role of HER3 in cetuximab resistance and the anti-tumor mechanisms of dual targeting of EGFR/HER3 in HNSCC. Materials and Methods: A cetuximab resistant HNSCC cell line SCC1-C, its parental cell line SCC1-P, and a HER3 knockdown counterpart SCC1-C/H were treated with cetuximab, MM121 and their combination. SRB and colony formation assays were performed to investigate the combined effect of MM121 and cetuximab on cancer cell growth. Activations of EGFR, HER2, HER3, AKT, and ERK signaling pathways were evaluated by Western blotting. The SCC1-C xenograft animal model and PDX models from 6 patients with HNSCC were utilized to determine the combined efficacy of MM121 and cetuximab. Results: Cetuximab induced HER3 activation and HER2/HER3 dimerization in HNSCC cell lines. The combined treatment blocked both EGFR and HER3 activation and inhibited both PI3K/AKT and ERK signaling pathways as well as HNSCC cell growth more effectively compared to either single antibody treatment in vitro. Furthermore, knock down of HER3 re-sensitized the resistant cell line to cetuximab. The anti-HER3/EGFR combination also demonstrated better efficacy than either single agent antibody in the cetuximab resistant HNSCC xenograft model and the PDX animal models. In PDX models from patients 1-4, both cetuximab and the combination significantly inhibited tumor growth in nude mice compare to the control (p&amp;lt;0.001 for both treatments) and there was no difference between single cetuximab and the combination. In 1 of the 4 PDXs, tumors treated with cetuximab grew back while no relapse could be detected in the combination group. In the PDX from patient 5, only the combination treatment significantly inhibited tumor growth (p&amp;lt;0.004) which was also more effective than cetuximab treatment (p&amp;lt;0.032). In the PDX from patient 6, both cetuximab and the combination significantly inhibited tumor growth in nude mice as compared with the control (p&amp;lt;0.001 for both treatments). However, the combination was significantly more effective than either of the single agents (p&amp;lt;0.0004 and p&amp;lt;0.003, respectively). Conclusion: Our study indicates that dual targeting of EGFR and HER3 is more effective than cetuximab alone in both cetuximab resistant HNSCC cell line and PDX models. These results pave the way for further clinical investigations using multiple targeting strategies in patients who have failed cetuximab based therapy. (The current study is supported by NIH grant R21 CA182661 to NFS and ZGC). Citation Format: Dongsheng Wang, Guoqing Qian, hongzheng zhang, kelly R. Magliocca, sreenivas nannapaneni, zhengjia chen, mihir R. patel, Mark W. El-Deiry, J.trad Wadsworth, dong M. Shin, fadlo R. khuri, Nabil F. Saba, Zhuo G. chen. HER3 targeting sensitizes HNSCC to cetuximab - evidence from cell line and patient derived xenograft (PDX) models. [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 1866.