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BACKGROUND: Plasticity of the ERBB receptor network has been suggested to cause acquired resistance to anti–human epidermal growth factor receptor 2 (HER2) therapies. Thus, we studied whether a novel approach using an ERBB1-3-neutralizing antibody mixture can block these compensatory mechanisms of resistance. METHODS: HER2+ cell lines and xenografts (n ≥ 6 mice per group) were treated with the ERBB1-3 antibody mixture Pan-HER, trastuzumab/lapatinib (TL), trastuzumab/pertuzumab (TP), or T-DM1. Downregulation of ERBB receptors was assessed by immunoblot analysis and immunohistochemistry. Paired pre- and post-T-DM1 tumor biopsies from patients (n = 11) with HER2-amplified breast cancer were evaluated for HER2 and P-HER3 expression by immunohistochemistry and/or fluorescence in situ hybridization. ERBB ligands were measured by quantitative reverse transcription polymerase chain reaction. Drug-resistant cells were generated by chronic treatment with T-DM1. All statistical tests were two-sided. RESULTS: Treatment with Pan-HER inhibited growth and promoted degradation of ERBB1-3 receptors in a panel of HER2+ breast cancer cells. Compared with TL, TP, and T-DM1, Pan-HER induced a similar antitumor effect against established BT474 and HCC1954 tumors, but was superior to TL against MDA-361 xenografts (TL mean = 2026 mm(3), SD = 924 mm(3), vs Pan-HER mean = 565 mm(3), SD = 499 mm(3), P = .04). Pan-HER-treated BT474 xenografts did not recur after treatment discontinuation, whereas tumors treated with TL, TP, and T-DM1 did. Post-TP and post-T-DM1 recurrent tumors expressed higher levels of neuregulin-1 (NRG1), HER3 and P-HER3 (all P < .05). Higher levels of P-HER3 protein and NRG1 mRNA were also observed in HER2+ breast cancers progressing after T-DM1 and trastuzumab (NRG1 transcript fold change ± SD; pretreatment = 2, SD = 1.9, vs post-treatment = 11.4, SD = 10.3, P = .04). The HER3-neutralizing antibody LJM716 resensitized the drug-resistant cells to T-DM1, suggesting a causal association between the NRG1-HER3 axis and drug resistance. Finally, Pan-HER treatment inhibited growth of HR6 trastuzumab- and T-DM1-resistant xenografts. CONCLUSIONS: These data suggest that upregulation of a NRG1-HER3 axis can mediate escape from anti-HER2 therapies. Further, multitargeted antibody mixtures, such as Pan-HER, can simultaneously remove and/or block targeted ERBB receptor and ligands, thus representing an effective approach against drug-sensitive and -resistant HER2+ cancers.

The ErbB receptor family member ErbB3 has been implicated in breast cancer growth, but it has yet to be determined whether its disruption is therapeutically valuable. In a mouse model of mammary carcinoma driven by the polyomavirus middle T (PyVmT) oncogene, the ErbB2 tyrosine kinase inhibitor lapatinib reduced the activation of ErbB3 and Akt as well as tumor cell growth. In this phosphatidylinositol-3 kinase (PI3K)-dependent tumor model, ErbB2 is part of a complex containing PyVmT, p85 (PI3K), and ErbB3, that is disrupted by treatment with lapatinib. Thus, full engagement of PI3K/Akt by ErbB2 in this oncogene-induced mouse tumor model may involve its ability to dimerize with and phosphorylate ErbB3, which itself directly binds PI3K. In this article, we report that ErbB3 is critical for PI3K/Akt-driven tumor formation triggered by the PyVmT oncogene. Tissue-specific, Cre-mediated deletion of ErbB3 reduced Akt phosphorylation, primary tumor growth, and pulmonary metastasis. Furthermore, EZN-3920, a chemically stabilized antisense oligonucleotide that targets the ErbB3 mRNA in vivo, produced similar effects while causing no toxicity in the mouse model. Our findings offer further preclinical evidence that ErbB3 ablation may be therapeutically effective in tumors where ErbB3 engages PI3K/Akt signaling. Cancer Res; 71(11); 3941–51. ©2011 AACR.

Topoisomerase I inhibitors down-regulate HIF-1α leading to tumor growth inhibition, but only while maintaining sustained levels of drug exposure. EZN-2208, a multi-arm 40 kDa pegylated, releasable SN38-drug conjugate, provides higher, longer lasting exposure of tumors to SN38 in contrast to SN38 that is released from CPT-11. EZN-2208 also consistently has greater antitumor activity than CPT-11 in a variety of solid and hematological tumor models. In this report, the ability of PEG-SN38 to down-regulate HIF-1α and its downstream targets, in a more potent, sustained manner compared with CPT-11 was examined. To do so, U251 glioma xenografts that stably expressed a hypoxia response element-dependent luciferase reporter gene were implanted in mice. After treatment it was found that EZN-2208 induced potent, sustained HIF-1α down-regulation (37% at 48 h and 83% at 120 h) in the tumors, whereas CPT-11 caused only minor, transient HIF-1α down-regulation. In addition, EZN-2208 down-regulated mRNA levels of HIF-1α targeted genes (MMP2, VEGF1, Glut1, Glut3 and TGFβ1). Further, western blot analyses of xenograft tumors demonstrated that EZN-2208 had significantly more effect than CPT-11 in down-regulating HIF-1α, VEGF, Glut1 and MMP2 protein levels. Significant down-regulation of HIF-1α and VEGF proteins translated to EZN-2208’s superior anti-angiogenic activity compared with CPT-11, confirmed by microvessel density reduction in a chorioallantoic membrane assay and in CD-31 immunohistochemistry studies. Additional studies done with matrigel implants devoid of tumor cells show that EZN-2208 significantly inhibits angiogenesis while CPT-11 has little or no effect. It is concluded that the superior antitumor activity of EZN-2208 compared with CPT-11 is attributed, in part, to an anti-angiogenic effect. Ongoing clinical Phase I and Phase II studies will assess safety and efficacy of EZN-2208. Electronic supplementary material The online version of this article (doi:10.1007/s10456-011-9209-1) contains supplementary material, which is available to authorized users.

496 Background: Sym004 consists of two anti-EGFR monoclonal antibodies (futuximab and modotuximab) directed against non-overlapping epitopes in the EGFR domain III. Sym004 induces rapid and efficient removal of the EGFR from the cancer cell surface by triggering EGFR internalization and degradation and has shown promising efficacy in mCRC patients. Based upon a post-hoc analysis of a Phase 2 study, a Phase 3 trial in genomically selected mCRC patients is in preparation. Methods: The aim was to establish a popPK model for Sym004 in order to i) evaluate impact of covariates (intrinsic and extrinsic factors) on Sym004 exposure and ii) provide exposure metrics for a PK/PD analysis. Sym004 serum concentrations were obtained from 330 patients with mCRC (n = 247) or advanced solid tumors (studies Sym004-01, Sym004-02, Sym004-05 and Sym004-06). Sym004 (0.4-18 mg/kg) was dosed by i.v. infusion weekly or every 2nd week, or as a 9 mg/kg loading dose followed by 6 mg/kg weekly (9/6 mg/kg weekly). Non-linear mixed effects modelling was done in NONMEM v7.3.0. Covariates evaluated included body weight, age, sex, race, albumin, renal function, hepatic function, tumor type and size, ECOG and previous anti-EGFR treatments. Results: The base popPK model was a 2-compartment model with linear and non-linear Michaelis-Menten-type elimination and a priori inclusion of body weight on CL, Vmax, V1 and V2. The model captured the non-linear PK well. The final covariate model retained covariates whose point estimates were outside the range of 0.8 to 1.25 and whose 90% confidence intervals did not overlap with the null value and included only body weight and albumin. Inter-individual variability was estimated for CL, Vmax and V1 and was in the range of 18-30%. Simulations were used to assess the clinical relevance of the covariates as judged by the magnitude of the change in exposure of the Phase 3 dose regimen of 9/6 mg/kg weekly. Conclusions: The popPK model described the Sym004 PK data well. No covariates were present that changed the Sym004 exposure in a clinically significant manner which would necessitate a dose modification. The model is suitable for simulating the Sym004 PK for PK/PD analyses. Clinical trial information: NCT01117428,NCT01417936,NCT02083653,NCT01955473.

Usually, small interfering RNAs and most antisense molecules need mechanical or chemical delivery methods to down-modulate the targeted mRNA. However, these delivery approaches complicate the interpretations of biological consequences. We show that locked nucleic acid (LNA)-based antisense oligonucleotides (LNA–ONs) readily down-modulate genes of interest in multiple cell lines without any delivery means. The down-modulation of genes was quick, robust, long-lasting and specific followed by potent down-modulation of protein. The efficiency of the effect varied among the 30 tumor cell lines investigated. The most robust effects were found in those cells where nuclear localization of the LNA–ON was clearly observed. Importantly, without using any delivery agent, we demonstrated that HER3 mRNA and protein could be efficiently down-modulated in cells and a tumor xenograft model. These data provide a simple and efficient approach to identify potential drug targets and animal models. Further elucidation of the mechanism of cellular uptake and trafficking of LNA–ONs may enhance not only the therapeutic values of this platform but also antisense molecules in general.

Immunotherapy has become a major focus of research in oncology and blockade of immune checkpoints such as cytotoxic T-Lymphocyte Associated Protein 4 (CTLA4) and programmed cell death protein 1 (PD1) has been some of the most successful immunotherapies. The next wave of immunomodulatory targets that are being explored for cancer therapy include T cell immunoglobulin and mucin domain protein 3 (TIM3). TIM3 is constitutively expressed on cells of myeloid origin whereas the TIM3 expression is induced on T-cells upon activation. The exact function of TIM3 on the different immune cells is not clear and may be context dependent suggesting that TIM3 is not a classical immune check-point. Sym023 is a human anti-TIM3 antibody, which binds human TIM3 and cross-reacts with cynomolgus monkey TIM3. Sym023 blocks binding of phosphatidyl serine but not galectin 9 and stimulates T-cell proliferation in mixed lymphocyte reactions and tumor growth inhibition in vivo. Here, we present data demonstrating that ligation of TIM3 by Sym023 increase cytokine production and T cell proliferation in vitro through a novel mechanism of action. Citation Format: Trine Lindsted, Monika Gad, Michael V. Grandal, Camilla Frölich, Vikram K. Bhatia, Torben Gjetting, Johan Lantto, Ivan D. Horak, Michael Kragh, Klaus Koefoed, Mikkel W. Pedersen. Preclinical characterization of Sym023 a human anti-TIM3 antibody with a novel mechanism of action [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 5629.

Inhibition of immunologic checkpoints like Programmed Cell Death 1 (PD-1) has shown clinical efficacy in a broad range of cancers by improving or restoring T-cell activity. Anti-PD-1 antibodies show great promise in treating cancer malignances when administered alone or in combination with other immune activating approaches. However, high protein sequence identity between human and mammalian species used for antibody generation often disfavor generation of antibodies against functionally conserved epitopes, or prevents isolating antibodies cross reacting with ortholog species used for evaluating potential toxicity. Chickens are phylogenetically distant from mammals and are better at generating antibodies against epitopes that are conserved in mammals. Because chickens generate antibodies from a very restricted set of V-gene germline genes that are diversified by “gene conversion”, we envisioned that high throughput humanization of antibody frameworks was achievable by “mass CDR grafting” after recovering antibodies by immunization and B-cell cloning. Wild type chickens were immunized with PD-1 antigen, and a repertoire of 120 antibodies was generated with Symplex™ technology, by combining single B-cell FACS sorting and high throughput RT-PCR cloning of cognate VH and VL chains. The isolated PD-1 repertoire was cloned with an inert Fc backbone and humanized by a combination of in silico CDR grafting and gene synthesis. Humanized antibodies were expressed and screened for retained binding affinity and functionality in T-cell based assays. We successfully generated a humanized PD-1 antibody repertoire and found that most antibodies retained affinity and functionality similar to that of parental chicken antibodies. Furthermore, the antibody repertoire displayed broad binding epitope coverage on PD-1, often with strong pM affinity, and showed biophysical properties acceptable for drug development. Our lead antibody Sym021 blocked PD-L1 and PD-L2 ligand binding and downstream PD-1 signaling, resulting in elevated T-cell cytokine production in vitro. Moreover, Sym021 bound human PD-1 with much stronger affinity of 30 pM compared to clinical PD-1 mAbs nivolumab and pembrolizumab, while also cross reacting to cynomolgous and mouse PD-1. This enabled direct testing of Sym021 in syngenic mouse in vivo models and evaluation of preclinical toxicology in cynomolgus monkeys. Detailed epitope mapping showed that the epitope recognized by Sym021 was unique compared to clinical antibodies pembrolizumab and nivolumab. These results supported entry of PD-1 targeting Sym021 into clinical trials. Citation Format: Torben Gjetting, Monika Gad, Camilla Fröhlich, Maria C. Melander, Gunther Galler, Johan Lantto, Thomas Bouquin, Ivan D. Horak, Michael Kragh, Mikkel W. Pedersen, Klaus Koefoed. Characterization of the first chicken-derived anti-PD-1 clinical stage antibody with a unique epitope and promising anticancer activity [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 3822.

Immunotherapy has become a major focus of research in oncology and blockade of immune checkpoints such as cytotoxic T-Lymphocyte associated protein 4 (CTLA4) and programmed cell death protein 1 (PD1) has been some of the most successful immunotherapies. Lymphocyte-activation gene 3 (LAG3) belongs to the second-generation immune modulatory targets. LAG3 is expressed by activated T-cells and tumor infiltrating lymphocytes (TILs) and negatively regulates T-cell activity upon ligand engagement. LAG3 binds major histocompatibility complex class II (MHCII) molecules found on the surface of antigen presenting cells and tumor cells. Sym022 is a Fc-inert human monoclonal antibody targeting LAG3. Sym022 binds to human and cynomolgus monkey LAG3 with high affinity and blocks the interaction between LAG3 and MHCII molecules. Functionally, Sym022 increases cytokine production by T-cells in vitro and tumor growth inhibition in vivo. Mechanistically, Sym022 not only blocks ligand binding, but also decreases total LAG3 surface levels through internalization and/or shedding. Citation Format: Michael M. Grandal, Maria C. Melander, Vikram K. Bhatia, Torben Gjetting, Trine Lindsted, Camilla Fröhlich, Johan Lantto, Ivan D. Horak, Michael Kragh, Klaus Kofoed, Mikkel W. Pedersen. Preclinical characterization of Sym022, a novel anti-LAG3 antibody [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 5626.

e15577Background: Sym004, an antibody mixture targeting EGFR, has demonstrated promising activity in patients (pts) with anti-EGFR refractory metastatic colorectal cancer (mCRC). A Phase 2b trial e...

Importance Acquired resistance to anti-EGFR therapy (epidermal growth factor receptor) is frequently due toRASandEGFRextracellular domain (ECD) mutations in metastatic colorectal cancer (mCRC). Some anti-EGFR–refractory patients retain tumor EGFR dependency potentially targetable by agents such as Sym004, which is a mixture of 2 nonoverlapping monoclonal antibodies targeting EGFR. Objective To determine if continuous blockade of EGFR by Sym004 has survival benefit. Design, Setting, and Participants Multicenter, phase 2, randomized, clinical trial comparing 2 regimens of Sym004 with investigator’s choice from March 6, 2014, through October 15, 2015. Circulating tumor DNA (ctDNA) was analyzed for biomarker and tracking clonal dynamics during treatment. Participants had wild-typeKRASexon 2 mCRC refractory to standard chemotherapy and acquired resistance to anti-EGFR monoclonal antibodies. Interventions Participants were randomly assigned in a 1:1:1 ratio to Sym004, 12 mg/kg/wk (arm A), Sym004, 9 mg/kg loading dose followed by 6 mg/kg/wk (arm B), or investigator’s choice of treatment (arm C). Main Outcomes and Measures Overall survival (OS). Secondary end points included preplanned exploratory biomarker analysis in ctDNA. Results A total of 254 patients were randomized (intent-to-treat [ITT] population) (median age, 63 [range, 34-91] years; 63% male; n = 160). Median OS in the ITT population was 7.9 months (95% CI, 6.5-9.9 months), 10.3 months (95% CI, 9.0-12.9 months), and 9.6 months (95% CI, 8.3-12.2 months) for arms A, B, and C, respectively (hazard ratio [HR], 1.31; 95% CI, 0.92-1.87 for A vs C; and HR, 0.97; 95% CI, 0.68-1.40 for B vs C). The ctDNA revealed high intrapatient genomic heterogeneity following anti-EGFR therapy. Sym004 effectively targetedEGFRECD-mutated cancer cells, and a decrease inEGFRECD ctDNA occurred in Sym004-treated patients. However, this did not translate into clinical benefit in patients withEGFRECD mutations, likely owing to co-occurring resistance mechanisms. A subgroup of patients was defined by ctDNA (RAS/BRAF/EGFRECD-mutation negative) associated with improved OS in Sym004-treated patients in arm B compared with arm C (median OS, 12.8 and 7.3 months, respectively). Conclusions and Relevance Sym004 did not improve OS in an unselected population of patients with mCRC and acquired anti-EGFR resistance. A prospective clinical validation of Sym004 efficacy in a ctDNA molecularly defined subgroup of patients with refractory mCRC is warranted. Trial Registration clinicaltrialsregister.eu Identifier:2013-003829-29

CPT-11, also known as irinotecan, is a prodrug that is approved for the treatment of advanced colorectal cancer. The active metabolite of CPT-11, SN38 (7-ethyl-10-hydroxy-camptothecin), has 100- to 1000-fold more potent cytotoxic activity in tissue cell culture compared with CPT-11. However, parental administration of SN38 is not possible because of its inherently poor water solubility. It is reported here that a multiarm poly(ethylene glycol) (PEG) backbone linked to four SN38 molecules (PEG-SN38) has been successfully prepared with high drug loading and significantly improved water solubility (400- to 1000-fold increase). Three different protecting strategies have been developed in order to selectively acylate the 20-OH of SN38 to preserve its E-ring in the lactone form (the active form of SN38 with cytotoxic activities) while PEG is still attached. One chemical process has been optimized to make a large quantity of the PEG-SN38 conjugate with a high yield that can be readily adapted for scale-up production. The PEG-SN38 conjugates have shown excellent in vitro anticancer activity, with potency similar to that of native SN38, in a panel of cancer cell lines. The PEG-SN38 conjugates also have demonstrated superior anticancer activity in the MX-1 xenograft mice model compared with CPT-11. Among the four conjugates, PEG-Gly-(20)-SN38 (23) has been selected as the lead candidate for further preclinical development.

The human epidermal growth factor receptor (HER)-family is involved in development of many epithelial cancers. Therefore, HER-family members constitute important targets for anti-cancer therapeutics such as monoclonal antibodies (mAbs). A limitation to the success of single HER-targeting mAbs is development of acquired resistance through mechanisms such as alterated receptor dimerization patterns and dependencies. Pan-HER is a mixture of six mAbs simultaneously targeting epidermal growth factor receptor (EGFR), HER2, and HER3 with two mAbs against each receptor. Pan-HER has previously demonstrated broader efficacy than targeting single or dual receptor combinations also in resistant settings. In light of this broad efficacy, we decided to investigate the effect of Pan-HER compared with single HER-targeting with single and dual mAbs on HER-family cross-talk and dimerization focusing on EGFR. The effect of Pan-HER on cell proliferation and HER-family receptor degradation was superior to treatment with single mAbs targeting either single receptor, and similar to targeting a single receptor with two non-overlapping antibodies. Furthermore, changes in EGFR-dimerization patterns after treatment with Pan-HER were investigated by in situ proximity ligation assay and co-immunoprecipitation, demonstrating that Pan-HER and the EGFR-targeting mAb mixture efficiently down-regulate basal EGFR homo- and heterodimerization in two tested cell lines, whereas single mAbs had limited effects. Pan-HER and the EGFR-targeting mAb mixture also blocked EGF-binding and thereby ligand-induced changes in EGFR-dimerization levels. These results suggest that Pan-HER reduces the cellular capability to switch HER-dependency and dimerization pattern in response to treatment and thus hold promise for future clinical development of Pan-HER in resistant settings. This article is protected by copyright. All rights reserved.

Purpose: Approved anti-EGFR antibodies cetuximab and panitumumab provide significant clinical benefit in patients with metastatic colorectal cancer (MCRC). However, patients ultimately develop disease progression, often driven by acquisition of mutations in the extracellular domain (ECD) of EGFR. Sym004 is a novel 1:1 mixture of two nonoverlapping anti-EGFR mAbs that recently showed promising clinical activity in a phase I trial in MCRC. Our aim was to determine the efficacy of Sym004 to circumvent cetuximab resistance driven by EGFR ECD mutations. Experimental Design: Functional studies were performed to assess drug–receptor binding as well as ligand-dependent activation of individual EGFR mutants in the presence of cetuximab, panitumumab, and Sym004. Cell viability and molecular effects of the drugs were assayed in cetuximab-resistant cell lines and in tumor xenograft models. Efficacy of Sym004 was evaluated in patients progressing to cetuximab that harbored EGFR mutation in the post-cetuximab tumor sample. Results: Contrary to cetuximab and panitumumab, Sym004 effectively bound and abrogated ligand-induced phosphorylation of all individual EGFR mutants. Cells resistant to cetuximab harboring mutations in EGFR maintained sensitivity to Sym004, which was consistent with an effective suppression of EGFR downstream signaling, translating into profound and sustained tumor regression in the xenograft model. As proof-of-principle, a patient with a tumor harboring an EGFR mutation (G465R) following cetuximab therapy benefited from Sym004 therapy. Conclusions: Sym004 is an active drug in MCRC resistant to cetuximab/panitumumab mediated by EGFR mutations. EGFR mutations are potential biomarkers of response to Sym004 to be evaluated in ongoing large clinical trials. Clin Cancer Res; 22(13); 3260–7. ©2016 AACR.

The human epidermal growth factor receptor (HER)-family is involved in development of many epithelial cancers. Therefore, HER-family members constitute important targets for anti-cancer therapeutics such as monoclonal antibodies (mAbs). A limitation to the success of single HER-targeting mAbs is development of acquired resistance through mechanisms such as alterted receptor dimerization patterns and dependencies. Pan-HER is a mixture of six mAbs simultaneously targeting epidermal growth factor receptor (EGFR), HER2 and HER3 with two mAbs against each receptor. Pan-HER has previously demonstrated broader efficacy than targeting single or dual receptor combinations also in resistant settings. In light of this broad efficacy, we decided to investigate the effect of Pan-HER compared with single HER-targeting with single and dual mAbs on HER-family cross-talk and dimerization focusing on EGFR. The effect of Pan-HER on cell proliferation and HER-family receptor degradation was superior to treatment with single mAbs targeting either single receptor, and similar to targeting a single receptor with two non-overlapping antibodies. Furthermore, changes in EGFR-dimerization patterns after treatment with Pan-HER were investigated by in situ proximity ligation assay and co-immunoprecipitation, demonstrating that Pan-HER and the EGFR-targeting mAb mixture efficiently down-regulate basal EGFR homo- and heterodimerization in two tested cell lines, whereas single mAbs had limited effects. Pan-HER and the EGFR-targeting mAb mixture also blocked EGF-binding and thereby ligand-induced changes in EGFR-dimerization levels. These results suggest that Pan-HER reduces the cellular capability to switch HER-dependency and dimerization pattern in response to treatment and thus hold promise for future clinical development of Pan-HER in resistant settings.

Tumor growth in the context of EGFR inhibitor resistance may remain EGFR-dependent and is mediated by mechanisms including compensatory ligand upregulation and de novo gene alterations. Sym004 is a two-antibody mixture targeting nonoverlapping EGFR epitopes. In preclinical models, Sym004 causes significant EGFR internalization and degradation, which translates into superior growth inhibition in the presence of ligands. In this phase I trial, we observed grade 3 skin toxicity and hypomagnesemia as mechanism-based dose-limiting events during dose escalation. In dose-expansion cohorts of 9 and 12 mg/kg of Sym004 weekly, patients with metastatic colorectal cancer and acquired EGFR inhibitor resistance were enrolled; 17 of 39 patients (44%) had tumor shrinkage, with 5 patients (13%) achieving partial response. Pharmacodynamic studies confirmed marked Sym004-induced EGFR downmodulation. MET gene amplification emerged in 1 patient during Sym004 treatment, and a partial response was seen in a patient with EGFRS492R mutation that is predictive of cetuximab resistance. Significance: Potent EGFR downmodulation with Sym004 in patients with metastatic colorectal cancer and acquired resistance to cetuximab/panitumumab translates into significant antitumor activity and validates the preclinical hypothesis that a proportion of tumors remains dependent on EGFR signaling. Further clinical development and expanded correlative analyses of response patterns with secondary RAS/EGFR mutations are warranted. Cancer Discov; 5(6);598–609. ©2015 AACR. See related commentary by Stintzing and Heinemann, p. 578 This article is highlighted in the In This Issue feature, p. 565

MET is a proto-oncogene that encodes a protein known as hepatocyte growth factor receptor (HGFR), involved in proliferative, survival and invasive/metastatic abilities of cancer cells. MET has received considerable attention as a potential target for cancer therapy, including gastric cancer (GC). MET amplification is present in 4-5% of GC patients, and associated with poor outcomes and significantly shorter median survival. Although intensive efforts have been directed toward the development of HGF-MET axis inhibitors, there are some issues for clinical translation; 1) proper biomarker to select patients, 2) proper chemotherapy combination partner and 3) proper line of treatment. Therefore, exploring drug to inhibit HGF-MET axis is essential. Among them, Sym015 (Copenhagen, Demark), a mixture of two monoclonal antibodies directed at MET receptor, is one of the leading agents in the pipeline. In this study, we had 49 GC cell lines including 27 Korean cancer patients. Those cells were analyzed by whole exome sequencing (WES) and RNA sequencing to understand biological and molecular characteristics. Also, expression levels of MET and MET-associated molecules were determined by western blot and HGF expression was evaluated by ELISA. Sensitivity of Sym015 was screened in 49 GC cell lines and combination with paclitaxel was performed in 6 MET amplification cell lines by CCK-8 assay. Combination index (CI) and dose reduction index (DRI) were evaluated by CalcuSyn software. Among 49 GC cell lines, we confirmed 6 cell lines with MET amplification including 2 novel Korean cancer patient cell lines (YCC-31 and YCC-34) and 17 (34.7 %) cell lines were sensitive to Sym015 with 20~68 % inhibition rate at 100 nM. And our data showed that Sym015 was related to MET amplification, c-Met/p-Met overexpression and exon 14 deletion, as we expected. Also, the 6 MET amplified cell lines (SNU5, SNU520, MKN45, YCC-31, YCC-34 and Hs746T) were all sensitive to Sym015 and down-regulate expression of c-Met/p-Met. Using MET amplified GC cell lines, we evaluated the efficacy and potential mechanism of Sym015 in combination with paclitaxel, which is the standard agent for second line treatment of metastatic GC. CI values determined at the ED50 indicated that 3 of 6 cell lines (MKN45, SNU520 and YCC-31) had synergistic effects (CI values < 0.7). When used in combination to treat 4 cell lines (MKN45, SNU520, YCC-31 and Hs-746T), the dose at ED50 of Sym015 could be reduced by 2.2-13.6 fold, based on DRI analysis. Also, the DRI for paclitaxel indicated that dose reductions up to 4.7-7.1 fold (MKN45 and SNU520) could be obtained. Our results indicate that Sym015 was sensitive to MET amplified cell lines and the Sym015 combined with paclitaxel therapy had synergistic effects in MET amplified GC. Citation Format: Hyun Jeong Kim, Sun Kyoung Kang, Ivan Horak, Michael Kragh, Woo Sun Kwon, Tae Soo Kim, Inhye Jeong, Sun Young Rha, Hyun Cheol Chung. Combination of paclitaxel and Sym015, a mixture of two monoclonal antibodies directed at MET receptor, to increase anti-tumor effects in gastric 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 27. doi:10.1158/1538-7445.AM2017-27

Aberrant over-activation of MET receptor tyrosine kinase is involved in driving malignancies such as gastric and non-small cell lung cancer (NSCLC), and in the development of resistance to EGFR-targeting therapeutics. This has led to the development of several MET-targeting agents in the form of tyrosine kinase inhibitors (TKIs) and monoclonal antibodies (mAbs), many of which are in clinical development. However, resistance to MET-targeted agents is an emerging problem. This study aims to understand mechanisms underlying resistance development to an analogue of emibetuzumab, a mAb targeting MET. Upon long term in vivo treatment, emibetuzumab-resistant tumors and cell lines were generated, isolated, and characterized to investigate their acquired resistance mechanisms. Extensive reverse phase protein array and network analysis were used to characterize the proteomic profiles of three resistant cell lines, revealing three distinct resistance profiles, one involving activation of the PI3Kinase/AKT/mTOR pathway. We further show, how these resistance mechanisms can be overcome by treatment with other targeting therapeutics both in vitro and in vivo. Two of the models demonstrated in vivo sensitivity to Sym015, a novel mixture of two mAbs targeting non-overlapping epitopes of MET, partly due to ADCC, indicating that Sym015 can overcome acquired resistance to emibetuzumab. The third model demonstrated a marked increase in PI3Kinase/AKT/mTOR pathway activation. This activation translated into induced cancer cell and tumor growth, which could be inhibited by agents targeting PI3Kinase, AKT, or mTOR. This study thus points to treatment of patients with acquired resistance to single targeting MET mAbs with PI3Kinase/AKT/mTOR-targeting agents or a MET-targeting antibody mixture such as Sym015. Citation Format: Sofie Ellebæk Pollmann, Emanuel Frank Petricoin, Valerie Calvert, Shruti Rao, Simina Boca, Subha Madhavan, Ivan David Horak, Andreas Kjær, Michael Kragh, Thomas Tuxen Poulsen. In vivo acquired resistance to an emibetuzumab analogue in MET-amplified gastric xenografts can be overcome by a MET-targeting antibody mixture or PIK3CA/AKT/mTOR inhibition [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 3158. doi:10.1158/1538-7445.AM2017-3158

Purpose: Accumulating evidence indicates a high degree of plasticity and compensatory signaling within the human epidermal growth factor receptor (HER) family, leading to resistance upon therapeutic intervention with HER family members. Experimental Design/Results: We have generated Pan-HER, a mixture of six antibodies targeting each of the HER family members EGFR, HER2, and HER3 with synergistic pairs of antibodies, which simultaneously remove all three targets, thereby preventing compensatory tumor promoting mechanisms within the HER family. Pan-HER induces potent growth inhibition in a range of cancer cell lines and xenograft models, including cell lines with acquired resistance to therapeutic antibodies. Pan-HER is also highly efficacious in the presence of HER family ligands, indicating that it is capable of overcoming acquired resistance due to increased ligand production. All three target specificities contribute to the enhanced efficacy, demonstrating a distinct benefit of combined HER family targeting when compared with single-receptor targeting. Conclusions: Our data show that simultaneous targeting of three receptors provides broader efficacy than targeting a single receptor or any combination of two receptors in the HER family, especially in the presence of HER family ligands. Pan-HER represents a novel strategy to deal with primary and acquired resistance due to tumor heterogeneity and plasticity in terms of HER family dependency and as such may be a viable alternative in the clinic. Clin Cancer Res; 21(18); 4110–22. ©2015 AACR. See related commentary by Yarden and Sela, p. 4030

Geldanamycin belongs to the family of benzoquinoid ansamycin tyrosine-kinase inhibitors. We have examined its effects on Her-2/neu kinase activity, protein expression level, and proliferation of Her-2+ malignant cells. In SK-BR-3 breast-cancer cells, short-time treatment with geldanamycin completely abrogated gp30-ligand-induced activation of Her-2 without a change of receptor-expression level. Longer treatment of intact cells with geldanamycin induced decreased steady-state Her-2 autophosphorylation activity, which correlated with reduction of Her-2 protein expression and phosphotyrosine content of several proteins. The decrease was time- and dose-dependent, starting after 1 hr at 100 nM concentration and reaching completion by 24 hr. The reduction of the Her-2 protein level probably resulted from increased degradation, since the Her-2 mRNA level remained constant. Geldanamycin effects were not specific for Her-2, since the non-receptor tyrosine-kinase fyn was inhibited equally. In contrast to these results, protein-kinase-C activity was not affected. In 3 other malignant cell lines expressing different amounts of Her-2 (SK-BR-3 > SK-OV-3 > OVCAR3 > MCF7), geldanamycin also effectively reduced Her-2-kinase activity proportionally to the decrease of protein expression. In contrast, in a [3H]-thymidine-uptake assay, cell growth was meaningfully inhibited by geldanamycin at nanomolar concentrations only in SK-BR-3 (IC50 2 nM) and MCF7 (IC50 20 nM), while OVCAR3 was only moderately sensitive (IC50 2 microM) and SK-OV-3 was clearly resistant to geldanamycin. In direct comparison with herbimycin A, another benzoquinoid ansamycin that has been more thoroughly characterized, the biologic effects of geldanamycin were more pronounced.

In attempt to elucidate the mechanism of the HIV infection induced T cell unresponsiveness, we studied signal-transducing molecules proximal to the T cell receptor (TCR) in T lymphocytes of HIV-infected individuals. Total amounts of protein tyrosine kinases (PTKs) Lck, Fyn, and ZAP-70 and the zeta chain of the TCR were found significantly decreased in T cells of symptomatic/AIDS patients as well as in T cells of individuals in acute and early asymptomatic stages of HIV infection. Unexpectedly, the detection of Lck, Fyn, and ZAP-70 was reversed after the treatment of cell lysates with dithiothreitol. This suggests that PTKs Lck, Fyn, and ZAP-70 were modified by a mechanism altering the status of sulfhydryl groups. Moreover, this mechanism seems to affect selectively T cells of HIV infected patients since B cell PTKs Syk and Lyn were detected structurally and functionally intact. Interestingly, similar alterations of signaling molecules were not detected in T cells of HIV-infected long-term asymptomatic individuals. Modification of T cell PTKs may thus underlie the HIV-induced impairment of lymphocyte function and may potentially predict disease progression.

Among the four human EGF receptor (HER) family members (EGFR, HER2, HER3, HER4), HER3 is of particular interest as it interacts with HER2 and EGFR via heterodimerization and is a key link to the phosphoinositide 3-kinase (PI3K)/AKT signal transduction axis. Recent studies indicate that HER3 plays a critical role in mediating resistance to agents that target EGFR or HER2. As HER3 lacks significant kinase activity and cannot be inhibited by tyrosine kinase inhibitors, neutralizing antibodies and alternative inhibitors of HER3 have been sought as cancer therapeutics. We describe here a locked nucleic acid (LNA)-based HER3 antisense oligonucleotide, EZN-3920, that specifically downmodulated the expression of HER3, which was associated with growth inhibition. EZN-3920 effectively downmodulated HER3 expression, HER3-driven PI3K/AKT signaling pathway, and growth in tumors derived from BT474M1 breast and HCC827 lung carcinoma cell lines, which overexpress HER2 and EGFR, respectively. Furthermore, when EZN-3920 was coadministered with gefitinib or lapatinib in xenograft tumor models, enhanced antitumor activity compared with the effect of monotherapy was found. The effect was associated with a blockade of induced HER3 mRNA expression caused by lapatinib or gefitinib treatment. Finally, EZN-3920 sustained its antiproliferative effect in trastuzumab-resistant cells and three independently derived gefitinib-resistant cells. Our findings show that downmodulation of HER3 by EZN-3920 leads to the suppression of tumor growth in vitro and in vivo, suggesting that HER3 can be an effective target for the treatment of various cancers that have been activated by HER3 alone or where HER3 activation is associated with EGFR or HER2 expression. Mol Cancer Ther; 12(4); 427–37. ©2013 AACR.

The tyrosine kinase receptor MET is involved in progression of a variety of human cancers and constitutes a promising therapeutic target. Particularly, subsets of tumors originating from lung or gastric tissues appear to be truly MET dependent. MET dependency is driven by alterations, such as MET-gene amplification, MET-exon 14 deletion, kinase activating mutations, or autocrine HGF production. Furthermore, MET-amplification has been reported as a key mechanism of de novo resistance to EGFR targeting agents in lung and colorectal cancers. Sym015, a novel antibody mixture comprising two monoclonal antibodies targeting non-overlapping epitopes on the SEMA domain of MET, was shown to effectively inhibit cell growth in vitro through effective MET degradation. In the present study, we screened a large panel of highly annotated human cancer cell lines for sensitivity to Sym015 in order to identify potential markers of response. Sym015 effectively inhibited growth of cell lines with MET-amplification, MET-exon 14 deletion, and autocrine HGF production, including MET-amplified cell lines with acquired resistance to EGFR targeting agents. To validate the in vitro findings, a range of cell line- and patient-derived xenograft models with MET amplification or Exon 14 deletion were tested for sensitivity to Sym015 and an analogue of the clinical stage anti-MET monoclonal antibody emibetuzumab (LY2875358). Sym015 effectively inhibited growth of tumors with autocrine HGF production, MET-amplification, and/or Exon 14 deletion, and had superior activity compared to the emibetuzumab analogue in many of the models. Importantly, tumors with a partial response to the emibetuzumab analogue were strongly inhibited by subsequent treatment with Sym015 in two MET-amplified models, one of which also harbors a MET-exon 14 deletion. In summary, our findings demonstrate a potent antitumor effect of Sym015 in MET-dependent models. The data thus strongly support initiation of clinical trials for patients with MET-amplification and Exon 14 deletions. Citation Format: Thomas T. Poulsen, Michael M. Grandal, Helle J. Jacobsen, Dorte S. Hansen, Trine Lindsted, Mikkel W. Pedersen, Ivan D. Horak, Michael Kragh, Johan Lantto. Sym015, a novel antibody mixture targeting non-overlapping epitopes of MET, effectively inhibits growth of MET dependent tumors and overcomes resistance to a single monoclonal antibody. [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 1219.

The receptor tyrosine kinase MET (Hepatocyte Growth Factor Receptor, HGFR) has been associated with development and progression of a range of human tumors due to its regulation of cell proliferation, migration, invasion, and angiogenesis. A subset of human tumors, particularly of lung or gastric origin, appear to have a primary dependency on MET, which is driven by alterations, such as MET-gene amplification, MET-exon 14 deletion, activating mutations, or autocrine HGF production. Furthermore, MET-amplification has been reported as a key mechanism of de novo resistance to EGFR targeting agents. Sym015 is a mixture of two humanized monoclonal antibodies against non-overlapping epitopes on MET. The specific pair of antibodies was identified in a high-throughput cell based screen searching for antibody mixtures with superior growth inhibitory activity against four MET-dependent cell lines. Both antibodies bind the SEMA domain of MET with high affinity. Synergistic activity was confirmed by dose-response curves in several MET-dependent cell lines and cell line and patient-derived xenograft models. Mechanistically, Sym015 blocks HGF binding to MET, induces MET internalization and degradation effectively diminishing MET oncogenic signaling. Sym015 also induces higher levels of antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro compared to individual mAbs. In conclusion, Sym015, a novel monoclonal antibody mixture against MET, shows strong inhibitory activity against MET driven cell lines and xenografts due to a combined effect of MET degradation and secondary effector functions. These data support initiation of clinical trials. Citation Format: Michael M. Grandal, Thomas T. Poulsen, Klaus Koefoed, Karsten W. Eriksen, Anna Dahlman, Paolo Conrotto, Thomas Bouquin, Helle Jacobsen, Ivan D. Horak, Michael Kragh, Johan Lantto, Mikkel W. Pedersen. A novel synergistic antibody pair targeting non-overlapping epitopes of MET effectively inhibits MET-driven cancer 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 1218.

The anti-EGFR monoclonal antibodies (MoAbs) cetuximab and panitumumab are used to treat ‘RAS’ wild type metastatic colorectal cancer (MCRC), providing significant clinical benefit in patients. However, all patients ultimately develop disease progression, driven by acquisition of mutations in downstream effectors and in the extracellular domain (ECD) of EGFR, in approximately 25% of the cases. Sym004 is a novel 1:1 mixture of two non-overlapping anti-EGFR MoAbs that has recently shown promising clinical activity in a phase I trial in MCRC. Our aim was to determine the efficacy of Sym004 to circumvent cetuximab resistance driven by EGFR ECD mutations. Functional studies were performed to assess drug-receptor binding as well as ligand-dependent activation of individual EGFR mutants in the presence of cetuximab, panitumumab and Sym004. Cell viability and molecular effects of the drugs were assayed in cetuximab-resistant cell lines and in tumor xenograft models. Efficacy of Sym004 was evaluated in patients progressing to cetuximab that harbored an EGFR mutation in the post-cetuximab tumor sample. Sym004 effectively bound and abrogated ligand-induced phosphorylation of all individual EGFR mutants. Cells resistant to cetuximab harboring EGFR ECD mutations maintained sensitivity to Sym004, which was consistent with an effective suppression of EGFR downstream signaling, translating into profound and sustained tumor regression in the xenograft model. As a proof of principle, a patient with a tumor harboring an EGFR mutation (G465R) following cetuximab therapy benefited from Sym004 therapy. These data suggest that Sym004 is an active drug in MCRC resistant to cetuximab/panitumumab mediated by EGFR mutations. Overall, EGFR mutations are potential biomarkers of response to Sym004 to be evaluated in ongoing large clinical trials. Citation Format: Francisco J. Sanchez-Martin, Alba Dalmases, Beatriz Bellosillo, Guillem Argiles, Mariona Gelabert, Israel Cañadas, Joana Vidal, Giulia Siravegna, Sabrina Arena, Klaus Koefoed, Laura Visa, Oriol Arpí, Ivan D. Horak, Mar Iglesias, Christopher Stroh, Michael Kragh, Ana Rovira, Joan Albanell, Alberto Bardelli, Josep Tabernero, Clara Montagut. The anti-EGFR antibody mixture Sym004 overcomes acquired resistance to cetuximab in colorectal cancer. [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 2149.

Bacterial lipopolysaccharide (LPS) induces a pleiotropic activation of the immune system which might subsequently result in septic shock. One of the cell surface receptors for LPS is the glycophosphatidylinositol-anchored protein CD14. Binding of LPS to CD14 induces production of lymphokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), IL-6, and IL-8, and CD14 is subsequently released from the cell surface. However, the mechanism of signaling via CD14 is still not known. We report here that protein tyrosine kinase (PTK) p56lyn is coupled to the LPS receptor CD14 in human monocytes. LPS rapidly activates CD14-associated p56lyn simultaneously with PTKs p58hck and p59c-fgr. Inhibition of PTKs by herbimycin A completely blocks LPS-induced down-modulation of CD14 and production of TNF-alpha and IL-1. These data suggest a critical role of PTKs in the LPS/CD14-mediated signal transduction pathway in human monocytes.

Adult T-cell leukemia (ATL) is a malignancy of mature lymphocytes caused by the retrovirus human T-cell lymphotrophic virus-I (HTLV-I). It is an aggressive leukemia with an overall mortality rate of 50% within 5 months; no conventional chemotherapy regimen appears successful in inducing long-term disease-free survival in ATL patients. However, ATL cells constitutively express high-affinity interleukin-2 receptors (IL-2Rs) identified by the anti-Tac monoclonal antibody, whereas normal resting cells do not. To exploit this difference in receptor expression, we administered anti-Tac intravenously (IV) to 19 patients with ATL. In general the patients did not suffer untoward reactions, and in 18 of 19 cases did not have a reduction in normal formed elements of the blood. Seven patients developed remissions that were mixed (1 patient), partial (4 patients), or complete (2 patients), with partial and complete remissions lasting from 9 weeks to more than 3 years as assessed by routine hematologic tests, immunofluorescence analysis, and molecular genetic analysis of T-cell receptor gene rearrangements and of HTLV-I proviral integration. Furthermore, remission was associated with a return to normal serum calcium levels and an improvement of liver function tests. Remission was also associated in some cases with an amelioration of the profound immunodeficiency state that characterizes ATL. Thus the use of a monoclonal antibody that blocks the interaction of IL-2 with its receptor expressed on ATL cells provides a rational approach for treatment of this aggressive malignancy.

Purpose: Treatment of neuroblastoma is successful in less than half of patients with high-risk disease. The antitumor activity of a water soluble pegylated SN38 drug conjugate, EZN-2208, was compared with CPT-11 (a prodrug for SN38) in preclinical models of human neuroblastoma. Experimental Design: The in vitro cytotoxicity of EZN-2208 was tested by counting trypan blue dye– and Annexin V–positive cells, whereas its therapeutic efficacy was evaluated, in terms of survival, and antitumor and antiangiogenic activities, in s.c. luciferase-transfected, pseudometastatic, and orthotopic neuroblastoma animal models. Results: EZN-2208 was about 100-fold more potent than CPT-11 in vitro, by inducing apoptosis/necrosis and p53 expression and by reducing hypoxia-inducible factor (HIF)-1α/HIF-2α expression. EZN-2208 gave superior antitumor effects compared with CPT-11 in neuroblastoma xenografts. EZN-2208 treatment always resulted in lack of tumor detection at the end of trials whereas only small therapeutic effects were observed with CPT-11, as assessed by luciferase assay or tumor size, or even by staining histologic sections of tumors with antibodies recognizing neuroblastoma cells and cell proliferation. In a neuroblastoma model resistant to doxorubicin, cisplatin, vincristine, fenretinide, and topotecan, EZN-2208 induced 100% curability. It also blocked tumor relapse after topotecan-vincristine-doxorubicin combined treatment. Mechanistic experiments showed statistically significantly enhanced terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling and Histone H2ax staining as well as decreased vascular endothelial growth factor, CD31, matrix metalloproteinase (MMP)-2, and MMP-9 expression in tumors removed from EZN-2208–treated mice and radiating vessels invading the tumor implanted onto the chorioallantoic membranes. Conclusions: EZN-2208 should be considered a most promising novel antineuroblastoma agent. An ongoing phase I study in pediatric patients should identify the optimal dose for a phase II study. Clin Cancer Res; 16(19); 4809–21. ©2010 AACR.

Survivin plays an important role in preventing apoptosis and permitting mitosis, and is highly expressed in various human cancers. EZN-3042 is a locked nucleic acid antisense oligonucleotide (LNA-AsODN) against survivin. We report the effects of EZN-3042 in animal models. In a chemical-induced liver regeneration model, treatment with a mouse homolog of EZN-3042 resulted in 80% down-modulation of survivin mRNA. In A549 and Calu-6 lung xenograft models, treatment with EZN-3042 single agent induced 60% down-modulation of survivin mRNA in tumors and 37-45% tumor growth inhibition (TGI). In Calu-6 model, when EZN-3042 was combined with paclitaxel, an 83% TGI was obtained. EZN-3042 is currently being evaluated in a Phase 1 clinical trial as a single agent and in combination with docetaxel.

Aryl-substituted amides of isomeric 6,7- and 7,8-dihydroxyisoquinoline-3-carboxamides (2 and 3)_ were prepared. Divergent structural requirements were observed for inhibiting p56lck and epidermal growth factor receptor (EGFR) protein-tyrosine kinases (PTKs), with the 7,8-dihydroxy substitution pattern being essential for p56lck activity, and the arylamide being required for EGFR potency. The work presents a useful approach toward the design of inhibitors which can discriminate between different PTKs.

Six patients with malignant lymphoma of lymphoblastic type involving cutaneous sites at time of diagnosis are presented. Skin sites of the head and neck were involved in all patients and included the scalp (three patients), forehead (two patients), and malar region of the face (one patient). Two patients also had additional sites of skin disease (neck, breast, and anterior trunk). In two patients the skin was the predominant site of disease, whereas in the remaining patients staging workup revealed generalized lymphoma. The histologic findings in each patient were typical of lymphoblastic lymphoma; the neoplastic cells were small with blastic nuclear chromatin. In three patients the neoplastic cells were convoluted, and in three they were nonconvoluted. Immunophenotypically, four lymphomas were of pre-B cell type, and two lymphomas were of T cell type. There was no correlation between histologic features and the immunophenotype. Since the majority of lymphoblastic lymphomas are of T cell type, the predominance of pre-B cell tumors involving the skin may suggest that pre-B cell neoplasms have a predilection for cutaneous involvement. In further support of this hypothesis, both lymphomas that appear to have arisen in the skin had a pre-B cell immunophenotype.

The epidermal growth factor receptor (EGFR) plays an important role in the development and maintenance of the malignant phenotype of several human cancers. Therefore, it is a frequently pursued therapeutic target and several antibodies targeting EGFR have been clinically approved. However, the clinical responses to current anti-EGFR agents are often modest, indicating a need for improved therapeutics. Resistance mechanisms to anti-EGFR antibodies include ligand up-regulation, receptor tyrosine kinase plasticity, and mutations in downstream signaling molecules such as KRAS, BRAF, and PIK3CA. Here, we present data showing that a mixture of antibodies targeting HER3, EGFR, and IGF1R (HEI mixture) with high affinity ligand-blocking antibodies induced growth inhibition in a large panel of cancer cell lines. Synergies of simultaneous inhibition of the three targets were frequently seen in cell lines and xenograft tumor models. In particular, colorectal cancer cell lines were sensitive to the HEI mixture and superiority to cetuximab was seen in several cell lines. Surprisingly, the HEI mixture was effective at inhibiting the growth of KRAS and BRAF mutated cell lines, suggesting that the three receptor tyrosine kinases may still contribute to the malignant phenotype of tumors with these genomic alterations. Colorectal cancer is thought to be a ligand driven disease and hence the inhibitory activity of the HEI mixture was tested in the presence of EGFR, HER3 and IGF1R ligands and compared with the activity of cetuximab. As expected, the HEI mixture was able to block growth stimulation induced by all ligands, whereas heregulin and IGF1/IGF2 caused resistance to cetuximab. A clinically relevant subpopulation of colorectal cancer harbors amplification of IGF2, a major ligand for IGF1R, and hence should display sensitivity to IGF1R targeting. The HEI mixture was tested in three metastatic colorectal PDX models with IGF2 overexpression and induced tumor regression in all three models. In conclusion, our data indicate that simultaneous targeting of EGFR, HER3 and IGFR1 provides a broader efficacy than targeting a single receptor, and thereby provides a rationale for simultaneous targeting of the three receptors with a recombinant antibody mixture. Citation Format: Christina Egebjerg, Camilla Fröhlich, Karsten Wessel Eriksen, Thomas Tuxen Poulsen, Klaus Kofoed, Thomas Bouquin, Andrea Bretotti, Livio Trusolino, Ivan David Horak, Michael Kragh, Mikkel Wandahl Pedersen. Effective targeting of colorectal cancer with a recombinant antibody mixture against EGFR, HER3 and IGF1R. [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 1668. doi:10.1158/1538-7445.AM2015-1668

l-Asparaginase (l-ASP), a bacterial enzyme used since the 1970s to treat acute lymphoblastic leukemia, selectively starves cells that cannot synthesize sufficient asparagine for their own needs. Molecular profiling of the NCI-60 cancer cell lines using five different microarray platforms showed strong negative correlations of asparagine synthetase (ASNS) expression and DNA copy number with sensitivity to l-ASP in the leukemia and ovarian cancer cell subsets. To assess whether the ovarian relationship is causal, we used RNA interference to silence ASNS in three ovarian lines and observed 4- to 5-fold potentiation of sensitivity to l-ASP with two of the lines. For OVCAR-8, the line that expresses the least ASNS, the potentiation was >500-fold. Significantly, that potentiation was >700-fold in the multidrug-resistant derivative OVCAR-8/ADR, showing that the causal relationship between ASNS expression and l-ASP activity survives development of classical multidrug resistance. Tissue microarrays confirmed low ASNS expression in a subset of clinical ovarian cancers as well as other tumor types. Overall, this pharmacogenomic/pharmacoproteomic study suggests the use of l-ASP for treatment of a subset of ovarian cancers (and perhaps other tumor types), with ASNS as a biomarker for patient selection. [Mol Cancer Ther 2006;5(11):2613–23]

Radiolabeling of monoclonal antibodies (mAbs) with an intracellularly trapped form of (131)I (residualizing (131)I) involves radioiodinating a small molecular entity, conjugating it to the mAb, and purification. Column purifications are impractical during procedures involving multi-gigabecquerel levels of radioactivity. The goal of this study was to develop a simple, remote, "1-pot" method of radiolabeling and purification for the scaled-up radioiodination of a humanized anti-carcinoembryonic antigen (CEA) mAb, humanized MN-14 (hMN-14; labetuzumab), with an optimized residualizing (131)I moiety, (131)I-IMP-R4. IMP-R4 is MCC-Lys(MCC)-Lys(X)-d-Tyr-d-Lys(X)-OH, where MCC is 4-(N-maleimidomethyl)-cyclohexane-1-carbonyl and X is 1-((4-thiocarbonylamino)benzyl)-diethylenetriaminepentaacetic acid.An IODO-GEN-based remote labeling system was used. IMP-R4 was radioiodinated (0.13 mumol per 3.7 GBq of (131)I) at a pH of 7.0-7.4 and conjugated to disulfide-reduced hMN-14 after quenching of unused reactive (131)I. The product was purified by stirring for 5 min with a 20% (w/v) suspension of an anion-exchange resin and sterilely filtered into a sealed vial. Human serum albumin was added at a final concentration of 1%-2.5%. Immunoreactivity was determined by mixing with CEA and determining the complexation level by size-exclusion high-pressure liquid chromatography. Two control radiolabelings, either with unreduced hMN-14 or with IMP-R4 omitted, also were performed.In 18 radiolabelings with (131)I in the range of 2.04-4.81 GBq (55-130 mCi), yields of 59.9% +/- 7.9% (mean +/- SD) at specific activities of 200 +/- 26 MBq/mg (5.4 +/- 0.7 mCi/mg) were obtained, withor =95% of the radioactivity being associated with hMN-14 and withor =4% aggregation. Similar yields were obtained in a subset of radiolabelings (n = 7) with3.7 GBq of (131)I. The immunoreactivities of the preparations were typically95%. Nonspecific incorporation in the absence of IMP-R4 was 0.5%, whereas that obtained with unreduced IgG was approximately 8%, possibly because of conjugation of IMP-R4 at lysine sites. The process also removed99% of the quenching reagent used. Radiolabelings performed with freshly prepared solutions or lyophilized preparations produced similar yields, a result that suggested the option for a single-use kit design.Efficient removal of (131)I-IMP-R4 and quenched (131)I by 5 min of stirring with anion-exchange resin renders a multi-gigabecquerel-level preparation of (131)I-IMP-R4-hMN-14 safe, convenient, and practical.

The aim of this work was to develop reagents and methods potentially useful in PET, using (68)Ga in a 2-step pretargeting protocol.We prepared bispecific antibodies (bsAbs) for disease-specific targeting of carcinoembryonic antigen-positive cells and recognition of later-administered bivalent hapten-peptide conjugates. The secondary antibody arm (antibody 679) recognizes a histaminyl-succinyl-glycine (HSG) structural subunit. The bsAbs were prepared as Fab' x Fab' conjugates using chemical cross-linking methods and as bispecific diabodies using recombinant DNA technologies. A HSG-bivalent hapten conjugate bearing the macrocyclic ring chelating agent 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) was designed to be readily radiolabeled with (68)Ga taken directly from a (68)Ge/(68)Ga generator system. Reagents were tested in vitro and, then, for their targeting properties in a preclinical animal model of human cancer.A chemically cross-linked hMN-14 x 679 F(ab')(2) and a fully humanized bispecific diabody construct (BS1.5H), expressed in Escherichia coli, were prepared for this work. We synthesized the bivalent peptide termed IMP 241 [DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH(2)] and labeled it with (68)Ga and (67)Ga at temperatures from 45 degrees C to 100 degrees C, over times of 15 min to 1 h, establishing 15 min at 95 degrees C as a useful condition for (68)Ga labeling. When we formulated the IMP 241 bivalent hapten-peptide with ammonium acetate buffer at pH 4-5 and eluted the (68)Ga from the generator directly into the peptide solution, we achieved an almost quantitative incorporation of the (68)Ga into IMP 241, as analyzed by size-exclusion high-performance liquid chromatography, after mixing the complex with the 679 antibody. For in vivo studies we used (67)Ga-IMP 241 as a surrogate for (68)Ga-IMP 241, in view of the short, 68-min half-life of the (68)Ga nuclide. The (67)Ga-IMP 241 was successfully pretargeted to human colon tumor xenografts in athymic mice with both the chemical and the diabody bispecific proteins. High tumor-to-normal tissue ratios for (67)Ga uptake were found for all tissues at 1 to 6 h after injection of (67)Ga-IMP 241. When using the BS1.5H diabody for pretargeting, tumor-to-blood, tumor-to-liver, and tumor-to-lung ratios of (67)Ga-IMP 241 at 1 and 3 h after injection were 41:1 and 137:1, 51:1 and 106:1, and 16:1 and 46:1, respectively.The general approach described, along with the new compositions and the labeling methods we have developed, may eventually allow for use of (68)Ga-labeled specific targeting agents in a routine clinical PET application.

The purpose of this research was to test the therapeutic efficacy of an anthracycline-antibody conjugate for the treatment of human B-cell lymphoma in a preclinical animal model.Doxorubicin (dox) conjugates of the murine and humanized versions of the anti B-cell antibody LL1, targeting CD74, were prepared, along with a nonspecific control dox-antibody conjugate, targeting carcinoembryonic antigen. Antibody conjugates carried approximately 8-10 drug molecules attached site-specifically at thiols of reduced interchain disulfide bonds. Conjugates were tested, initially in vitro, and then for therapeutic efficacy in a systemic model, using a lethal i.v. dose of Raji cells in SCID mice.Dox-LL1 conjugates were shown to be stable and 3-fold more effective in vitro against the human B-cell Burkitt's lymphoma line, Raji, compared with the nonspecific control conjugate that did not target CD74 or B cells. When SCID mice were given an i.v. dose of 2.5 million Raji cells, they would die of disseminated disease within 15-25 days postinjection. A single dose of dox-LL1 conjugate, 117-350 micro g, given 5 days to 14 (advanced disease) days after injection of the Raji cells resulted in cure of most animals out to 180 days after injection of the cells, whereas animals in treatment control groups were not cured. The dose of dox-LL1 found useful in this work corresponds with a significantly lower drug dose than reported previously with other drug-antibody conjugatesCD74 appears to be a uniquely useful target antigen for delivery of drugs, effecting cures of animals with single, low doses of conjugate.

The inhibition of cellular processes associated with the malignant pheonotype is the goal many oncolytics. In the antisense oligonucleotide (ASO) approach, Watson and Crick base-pairing rules serve as the basis of rational drug design to create single-stranded oligonucleotides that are complementary and bind to RNAs critical for the malignant phenotype. The targets for antisense approaches can be mRNAs that encode for disease related proteins like those that control tumor growth, cell division, or survival. More recently, RNA targets in oncology have been expanded to include microRNAs. A successful oncolytic oligonucleotide must effectively bind and inhibit a target mRNA or miRNA that is critical for the malignant transformation or survival. Target identification presents the same challenge in antisense therapeutics as any other class of oncolytics, but drug design is based the known sequence of the target RNA. The first clinical trials to use antisense oligonucleotides in oncology produced only modest efficacy in clinical trials, but the therapeutic potential of these initial antisense drugs may have been hampered by their low stability and low binding affinity. Advancements in oligonucleotide chemistry have produced oligonucleotides with increased stability and increased affinities. This talk will focus on the use of oligonucleotides with the locked nucleic acid (LNA) modification. In these antisense constructs, some of the nucleotides in the sequence have a modification of the ribose sugar that includes a methylene bridge between the 2’ and 4’ positions. This bridge functions to lock the ribose into a (C3’-endo) configuration: a configuration that is optimized for binding to its cognate nucleotide. LNA-modified oligonucleotides bind to their target RNAs with higher affinities than most other oligonucleotide chemistries. One measure of affinity is the melting temperature (Tm) for binding to its complementary sequence. For each LNA-modified nucleotide added to an ASO sequence, Tm can increase Tm by over 5°. For example, an LNA-modified oligonucleotide currently in clinical development, miravirsen (SPC3649), with 9 LNA-modified nucleotides has a Tm of approximately 80° demonstrating that LNA-modified oligonucleotides have sufficient affinities to bind to and inhibit RNA function. Affinities like these have translated into increase potency for the inhibition of target RNAs in nonclinical models and should yield therapeutic benefit more robust than those seen with earlier generations of oligonucleotide therapeutics. Other drug-like properties of antisense therapeutics have also been improved by including LNA-modified nucleotides. For example, the pharmacokinetic properties of antisense oligonucleotides support their use in clinical medicine. Antisense oligonucleotides are single stranded and bind to proteins in circulation and on cell surfaces. After parenteral administration, antisense oligonucleotide are bound first to plasma proteins, limiting glomerular filtration, and then later the antisense oligonucleotides appear bound to cell surface (proteins), allowing them to be internalized in cells. Delivery into cells and tissues is achieved without the need for formulations more complex than just aqueous solutions. LNA modifications increase the metabolic stability in both circulation and inside of cells resulting in stable tissue concentrations and prolonged activities. This long tissue residence and stability, allows for constant exposure in tumor cells with dosing as infrequent as weekly or fortnightly. Again, this represents an improvement over antisense drugs used in previous oncology trials. Using in vitro and in vivo laboratory models it has been possible to demonstrate that treatment with LNA-modified oligonucleotides produces reductions in target gene expression and ultimately tumor growth that are sequence-, concentration-, and duration-of-therapy-dependent. Taken together the properties of LNA-modified oligonucleotides make them excellent candidates for inhibiting key RNA targets and at this time there are multiple LNA-modified oligonucleotides in clinical trials in oncology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY31-02. doi:10.1158/1538-7445.AM2011-SY31-02

Sym013 represents a novel HER family targeting approach consisting of a mixture of six monoclonal antibodies against EGFR, HER2, and HER3. Sym013 effectively induces rapid, simultaneous down-regulation of all HER members. Compared with single receptor or dual receptor targeting of the HER family, Sym013 provides broader inhibition and greater down-regulation efficacy. In this current study, we examined the capacity of Sym013 in combination with ionizing radiation to augment tumor response in lung (NSCLC) and head and neck (HNSCC) cancer model systems. The anti-proliferative effects of Sym013 were confirmed showing 30-60% growth inhibition across a variety of NSCLC and H&N cancer cell lines that express EGFR, HER2, and/or HER3. Furthermore, Sym013 inhibited the growth of tumor cells that exhibit resistance to cetuximab. Western blot analysis showed a dose-dependent down-regulation of HER family members and downstream MAPK and AKT signaling pathways following 24-hour treatment with Sym013. Clonogenic survival analysis revealed that Sym013 enhanced radiosensitivity in tumor cells following radiation exposure. We characterized the DNA damage profile following radiation via flow cytometric analysis of phosphorylated histone 2AX (γH2AX) foci. This revealed a significant increase of γH2AX following treatment with Sym013 and 2 Gy radiation when compared with Sym013 or radiation alone. Furthermore, combined treatment of Sym013 and radiation induced a significant G1 and G2 arrest. Using Annexin V/PI staining, we also found a significant increase in the apoptotic cell population following treatment of Sym013 and 6 Gy radiation. These results suggest that Sym013 augments radiation response via the induction of cell cycle arrest followed by the induction of apoptosis and cell death, likely reflecting inhibitory effects on DNA damage repair. Additional studies to examine the impact of Sym013 to augment radiation response as well as overcome acquired resistance to cetuximab in xenograft models are in progress. Overall, these data suggest the significant capacity of Sym013 to augment radiation response in lung and head and neck cancers. This unique antibody mixture warrants additional investigation as a promising novel HER family targeting strategy in cancer therapy. Citation Format: David Francis, Shyhmin Huang, Lauryn Werner, Johan Lantto, Ivan D. Horak, Michael Kragh, Paul M. Harari. Sym013, novel pan-HER monoclonal antibody mixture, augments radiation response in human lung and head and neck tumors. [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 4495. doi:10.1158/1538-7445.AM2014-4495

Deregulation of HER family receptors plays an important role in the development and progression of human cancer. Although EGFR and HER2 receptors have proven to be clinically valuable targets, increasing evidence suggests substantial cross-talk between HER family members, and thus compensatory signaling may lead to resistance upon therapies targeting one receptor only. Targeting multiple receptors is more efficacious than single-targeted therapies, and is frequently able to overcome acquired resistance to inhibition of a single receptor. We have previously demonstrated that Pan-HER (Sym013) a mixture of monoclonal antibodies specifically targeting EGFR, HER2, and HER3 displays potent growth inhibition in a broad range of cancer cell lines. Growth-inhibiting activity persisted in the presence of EGFR and HER3 ligands, indicating that Pan-HER may overcome acquired resistance due to increased ligand production. In vitro findings further translated into in vivo model systems, where Pan-HER displayed efficacious tumor growth suppression across a broad range of cancer models, including KRAS-mutated patient-derived pancreatic cancer models, and was frequently able to overcome resistance to other HER family targeted therapies. Mechanistic studies confirm that Pan-HER treatment significantly decreases viability and induces apoptosis more effectively than single-receptor targeted therapies. Pan-HER treatment induces internalization and degradation of all three receptors in vitro, followed by subsequent downstream effects in essential growth and survival pathways. Compensatory receptor up-regulation and/or activation is frequently the downside of single-receptor targeted treatments. However, such receptor up-regulation is effectively prevented by Pan-HER. Histological assessment of Pan-HER-treated patient-derived xenograft tumors revealed that they largely consist of stromal cells, whereas tumor cells are sparse. Further immunohistochemical staining has also shown that these tumors have markedly lower EGFR, HER2, and HER3 receptor expression than tumors treated with single-receptor targeting regimens. We present here further evidence of Pan-HER superiority over single-receptor targeted regimens. Overall, Pan-HER represents a novel strategy to address tumor heterogeneity andtumor plasticity. Citation Format: Anna Dahlman, Helle J. Jacobsen, Thomas T. Poulsen, Paolo Conrotto, Mikkel W. Pedersen, Ivan D. Horak, Michael Kragh, Johan Lantto. Simultaneous inhibition of HER-family receptors by Pan-HER antibody mixture prevents compensatory HER-family receptor upregulation and induces cell death in a broad range of tumor models. [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 657. doi:10.1158/1538-7445.AM2014-657

Dysregulation and overexpression of the human epidermal growth factor receptor (HER) family plays an important role in many cancers. Simultaneously targeting multiple members of the HER family may increase the therapeutic efficacy. Here, we report the ability to image the therapeutic response obtained by targeting the HER family members individually or simultaneously using novel monoclonal antibody (mAb) mixtures. Mice with subcutaneous BxPC-3 pancreas adenocarcinomas were divided into five groups (n=8) receiving vehicle or mAb mixtures directed against HER1 (EGFR), HER2, HER3, or pan-HER. Small animal positron emission tomography/computed tomography (PET/CT) with 18F-FDG and 18F-FLT was performed at baseline and day 1 or 2 after initiation of therapy. The changes in tumor uptake of tracers were quantified and related to the efficacy of each therapy on tumor size measured by caliper. Imaging results were further validated by immunohistochemistry on tumor samples obtained 2 and 7 days after initiation of therapy. The pan-HER mAb mixture showed the greatest efficacy with significant smaller tumor volumes (146±39 mm3, Mean ±SEM) compared with the other groups (vehicle: 660±99 mm3, EGFR: 363±67 mm3, HER2: 458±45 mm3, HER3: 274±37 mm3) at the end of therapy (day 23). Mean FDG and FLT uptake in the pan-HER group decreased by 19±4.3% and 24±3.1%, respectively, at day 1 or 2 compared with baseline, which was significantly greater than for the other groups. Importantly, the early change in mean FDG and FLT uptake correlated with tumor growth at day 23 relative to day 0 (r=0.67, p In conclusion, we demonstrate the ability to image therapeutic responses to a novel pan-HER mAb mixture using PET/CT imaging with FDG and FLT. We suggest the use of FDG and FLT PET/CT as an imaging biomarker in clinical evaluation of the pan-HER mAb mixture. Citation Format: Carsten H. Nielsen, Mette M. Jensen, Helle J. Jacobsen, Thomas T. Poulsen, Ivan D. Horak, Johan Lantto, Michael Kragh, Andreas Kjaer. In vivo imaging of therapy response to novel antibody mixtures targeted at the human epidermal growth factor receptor family using FDG and FLT positron emission tomography. [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 2070. doi:10.1158/1538-7445.AM2014-2070

3551 Background: Preclinical models suggest that WT KRAS mCRC may retain EGFR dependency despite resistance developed to anti-EGFR mAb treatment (eg, cetuximab or panitumumab). Sym004 is the first-...

3551 Background: KRAS wt mCRC pts progressing on chemotherapy and anti-EGFR mAbs have limited treatment options. Sym004 is a first-in-class drug mixture of two mAbs targeting non-overlapping epitopes on the EGFR, causing its internalization and degradation. With this unique mechanism of action, Sym004 overcomes acquired resistance to anti-EGFR mAbs in preclinical studies. Methods: Open-label, multicenter trial assessing safety (primary endpoint) and efficacy of 2 dose levels of Sym004 in KRAS wt mCRC pts with prior clinical benefit to anti-EGFR mAbs and subsequent progression during or within 6 months after treatment cessation. Sym004 was administered until disease progression or unacceptable toxicity. Tumor responses were evaluated centrally according to RECIST criteria. Paired skin and tumor biopsies were obtained at baseline and week 4. Results: In total, 42 pts were enrolled at 9 mg/kg (13) and 12 mg/kg (29). Median age was 66 years and median number of prior treatment lines 3. Central radiology review was performed in 12/13 (92%) pts at 9 mg/kg and 27/29 (93%) pts at 12 mg/kg. Tumor shrinkage > 10% was documented in 4/12 (33%) pts at 9 mg/kg, with partial response (PR) in 1/12 (8%) and stable disease (SD) in 9/12 (75%). At 12 mg/kg, 7/27 (26%) pts had > 10% tumor shrinkage, with PR in 3/27 (11%) and SD in 15/27 (56%). Median progression-free survival was 13.6 weeks (95% CI: 5.3-23) and 13.7 weeks (95% CI: 5.9-18.6), respectively. Duration of response for pts with PR was 5.6-17.6 weeks. Grade 3 or higher toxicity included skin rash in 26/42 (62%), hypomagnesemia in 16/42 (38%) and diarrhea in 2/42 (8%). Adverse events were manageable with dose reduction and supportive medication. There were no indications of immunogenicity. Pharmacodynamic analysis in serial tumor samples showed profound down-regulation of EGFR and reduction in proliferation marker Ki67. Conclusions: Sym004 at weekly doses of 9 and 12 mg/kg showed significant clinical activity in anti-EGFR treatment-refractory KRAS wt mCRC pts, clearly demonstrating proof-of-concept. Serial biopsies confirmed its mechanism of action. No unexpected adverse events were observed. Clinical trial information: NCT01117428.

6002 Background: Sym004 is a first-in-class drug mixture of two mAbs targeting non-overlapping epitopes on the EGFR. In preclinical models, Sym004 exhibited more pronounced EGFR internalization, degradation and tumor growth inhibition than cetuximab. Sym004 was investigated as monotherapy in palliative squamous cell carcinoma of the head and neck (SCCHN) patients (pts). Methods: SCCHN pts progressing after anti-EGFR mAbs for palliation were eligible. Documented clinical benefit (PR, CR or SD for at least 8 weeks according to RECIST) on an anti-EGFR mAb-containing regimen followed by disease progression during or within 12 weeks after treatment cessation was required. The primary endpoint of this multicentre single arm trial was centrally evaluated progression-free survival (PFS), estimated by median PFS and 24 week progression free rate. Secondary endpoints included objective tumor response, safety, biomarkers and pharmacokinetics. Pts received weekly iv infusions of 12 mg/kg Sym004 until disease progression. Tumor evaluation was performed week 6, 12 and every 8 weeks thereafter. Results: Based on the statistical hypothesis, 26 pts were included, of whom 23 had progressed while on anti-EGFR mAb treatment. One of 19 evaluable pts was HPV positive and no EGFRvIII mutation was detected in 21 evaluable pts. No anti-drug antibodies were detected. Independent central review of CT/MRI scans from 20 evaluable pts showed tumor shrinkage in 8 pts (% decrease in sum of the largest diameters: 6.5, 7.1, 9.6, 10.2, 11.3, 13.6, 16.7, 27.1) and 14 pts had SD as best overall response. Median PFS was 82 days (95% CI: 41, 140) and 24 week progression free rate was 12% (95% CI: 1, 39). During treatment 25/26 (96%) pts developed skin rash with ≥ grade 3 reported in 11/26 (42%) pts. Hypomagnesemia ≥ grade 3 was reported in 10/26 (38%) pts. Sym004 treatment resulted in a marked down regulation of EGFR in centrally reviewed biopsies from skin and tumors. Conclusions: Sym004 demonstrated clinical activity in heavily pretreated, predominately HPV negative pts with advanced SCCHN previously progressing on or after anti-EGFR mAbs. No unexpected toxicities were reported. Clinical trial information: NCT01417936.

INTRODUCTION Glioblastoma (GBM) is the most common intracranial cancer in adults but despite recent advances in therapy the overall survival remains about 20 months. The epidermal growth factor receptor variant III (EGFRvIII) is a truncated, constitutively active, and highly oncogenic form of the EGFR expressed on approximately 30% of GBMs. Sym004 is a recombinant IgG1 antibody mixture consisting of two antibodies against domain III of the epidermal growth factor receptor (EGFR). Like anti-EGFR monoclonal antibodies, Sym004 inhibits cancer cell growth and survival by blocking ligand-binding and receptor signaling. However, unlike the monoclonal antibodies, Sym004 induces a rapid and efficient internalization and degradation of the EGFR. Here, we examine whether the more efficient removal of the EGFR and the constitutive active EGFRvIII by Sym004 would translate into increased tumor growth inhibition of EGFRvIII GBM xenografts. METHODS Both subcutaneous (sc) and intracranial (ic) adult brain tumor xenografts were grown in athymic BALB/c mice. After tumor size reached 200-500 mm3 subcutaneously or 3 days after intracranial implantation, groups of 10 mice were randomly treated with either drug vehicle, Sym004 (50 mg/kg) or cetuximab (50 mg/kg) IP twice weekly for 5 weeks. Tumor responses for sc xenografts were assessed by tumor growth delay and regression and for ic xenografts by difference in median survival. Xenograft lines utilized for this study expressed either primarily EGFR wildtype (D-54 MG) or mutant EGFRvIII (D-270 MG and D-317 MG). RESULTS AND DISCUSSION The sc results for D-54 MG did not demonstrate statistically significant growth delays for either Sym004 or cetuximab. In sc EGFRvIII models, however, Sym004 produced statistically significant (p CONCLUSIONS The novel strategy to target EGFR demonstrates the significant anti-tumor activity of Sym004 against adult glioblastoma xenografts that express EGFRvIII. These results warrant further exploration of Sym004 in clinical trials for the treatment of EGFRvIII positive brain tumors. Funding for these studies was provided by Symphogen and the Tisch Preclinical Therapy Screening Program Citation Format: Stephen T. Keir, Martin A. Roskoski, Michael Kragh, Mikkel W. Pederson, Helle J. Jacobsen, Ivan D. Horak, Henry S. Friedman, Darell D. Bigner. Efficacy of Sym004, a novel anti-EGFR antibody mixture, against EGFRvIII positive glioblastoma xenografts. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4322. doi:10.1158/1538-7445.AM2013-4322

EGFR mutant lung cancers are highly sensitive to first generation EGFR tyrosine kinase inhibitors (TKIs; gefitinib and erlotinib), but resistance invariably develops. In the majority of patients, such acquired resistance is mediated by a second-site T790M mutation in EGFR. Second generation EGFR TKIs, such as afatinib, have minimal efficacy in patients with acquired resistance; by contrast, combinations of afatinib with cetuximab or panitumumab - anti-EGFR monoclonal antibodies - are synergistic in pre-clinical models, and afatinib/cetuximab has shown a 30% response rate in a phase Ib trial for patients with acquired resistance. Sym004, currently in phase II trials, is a novel mixture of two anti-EGFR monoclonal antibodies binding non-overlapping epitopes in the extracellular domain III of 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. To determine if the combination of afatinib and Sym004 shows greater efficacy against TKI-resistant EGFR mutant lung cancer than afatinib combined with individual anti-EGFR monoclonal antibodies, we are investigating mechanisms of sensitivity and resistance to afatinib plus Sym004 in EGFR mutant TKI-resistant lung cancer. Similar to cetuximab, Sym004 has minimal effect on the growth of EGFR mutant cells in 2D culture. However, Sym004 induces growth inhibition of TKI-resistant PC-9/BRc1 cells (EGFR exon 19 deletion/T790M) in soft agar and xenograft models, and afatinib plus Sym004 is more effective at inhibiting growth than either drug alone. In immunoblotting studies, the combination induces greater decrease of total EGFR than either drug alone or afatinib plus cetuximab. Using Alexa Fluor 488-labeled Sym004 and confocal microscopy, the drug induces more efficient internalization and degradation of EGFR than cetuximab in H1975 cells (L858R/T790M). Additional confocal studies will be performed in other TKI-resistant EGFR mutant lung cancer cells. We are also assessing whether addition of Sym004 to TKIs can prevent or delay the acquisition of T790M-mediated resistance in vitro. Finally, we are developing cell lines from PC-9/BRc1and HCC827/R1 (exon 19 deletion/T790M) xenografts that develop resistance to either Sym004, cetuximab, or combinations with afatinib. Collectively, these studies could provide insights into the biology of EGFR mutant lung cancers and preclinical rationale for a trial with the combination treatment of afatinib and Sym004 in patients with EGFR mutant lung cancer and acquired resistance to TKIs. Citation Format: Catherine Meador, Kadoaki Ohashi, Yumei Pan, Elisa de Stanchina, Mikkel W. Pedersen, Ivan Horak, Michael Kragh, William Pao. Mechanisms of sensitivity and resistance to Sym004, a novel anti-EGFR antibody mixture, in EGFR mutant lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2438. doi:10.1158/1538-7445.AM2013-2438

Deregulation of HER family members plays an important role in the development and progression of human cancer, and EGFR and HER2 are also clinically validated targets in many cancer indications. Accumulating evidence indicates, however, a high degree of HER family cross-talk and compensatory signaling leading to resistance upon therapeutic intervention with one of the receptors. Simultaneous targeting of more than one HER family receptor in vivo is frequently able to overcome resistance to the initial drug and the combined treatment is often also more efficacious than single-receptor targeting alone. Blocking of signaling from more than one HER family member may thus be necessary to effectively treat cancer and limit drug resistance. We have generated Sym013, a mixture of monoclonal antibodies specifically targeting EGFR, HER2 and HER3. Preclinical testing has shown that Sym013 displays potent growth inhibitory activity in a broad range of cell lines of diverse tissue origin and genetic background, including cell lines with acquired resistance to cetuximab, trastuzumab, or pertuzumab. Sym013 effectively inhibits all three targets simultaneously and thus prevents compensatory receptor up-regulation/activation, a commonly observed cellular response that can lead to drug escape when a single receptor is targeted. Furthermore, Sym013 is highly efficacious in the presence of EGFR and HER3 ligands, indicating that it is capable of overcoming acquired resistance due to increased ligand production. Overall, simultaneous targeting of three receptors provides broader efficacy than targeting of a single receptor or any combination of two receptors in the HER family. The ability to simultaneously inhibit EGFR, HER2 and HER3 in cancer cells in vitro also translates into broad and efficacious tumor growth suppression in vivo. All three target specificities have been demonstrated to contribute to the efficacy of Sym013 in vivo, and there is a clear benefit of combining HER family target specificities compared to single-receptor targeting. Sym013 is also highly efficacious in hard-to-treat KRAS mutated patient-derived pancreatic cancer models, and is frequently able to overcome resistance to HER family targeted therapeutics. Our data suggest that a mixture of antibodies, which simultaneously inhibits EGFR, HER2 and HER3, is superior to existing targeted therapies in dealing with both primary and acquired resistance due to intra-tumor heterogeneity and plasticity in terms of HER family dependency. Citation Format: Johan Lantto, Mikkel W. Pedersen, Helle J. Jacobsen, Thomas T. Poulsen, Ida Kjær, Klaus Koefoed, Jette W. Sen, Dietmar Weilguny, Bolette Bjerregaard, Christina R. Andersen, Ivan D. Horak, Michael Kragh. Simultaneous inhibition of EGFR, HER2 and HER3 by an antibody mixture provides broad and potent tumor inhibition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4751. doi:10.1158/1538-7445.AM2013-4751