Preclinical Xenograft Models Research Articles

Abstract 4902: Celecoxib fixed-dose combination: antitumor activity on tumor growth and metastasis

Abstract Background: Breast cancer and melanoma are associated with a high rate of lymphatic and hematogenous metastasis which results in high rate of cancer mortality. Inflammation and hypertension (HTN) have recently emerged as causal factors for tumor progression and anti-hypertensive agents have been shown to reduce inflammation and suppress tumor growth and metastasis. Cyclooxygenase-2 (COX-2) is upregulated in most human tumors and is a potent inducer of cancer-associated inflammation that promotes tumor angiogenesis and lymphangiogenesis. This study evaluated a novel combination of a selective COX-2 inhibitor with three antihypertensive drugs to suppress tumor growth and metastasis in preclinical xenograft models. Methods: Three anti-hypertensive drugs were evaluated in this study: i) Lisinopril [LIS], an inhibitor of angiotensin-1 converting enzyme (ACE); ii) Olmesartan medoxomil (OLM), an angiotensin II receptor blocker (ARB); and iii) Hydrochlorothiazide (HCTZ), a thiazide diuretic along with Celecoxib [CEL], a selective COX-2 inhibitor. CEL, LIS, OLM, and HCTZ were evaluated either alone or in combination for tumor growth suppression and metastatic spread in an orthotopic xenograft model of triple-negative inflammatory breast cancer/SUM149 and subcutaneous xenograft models of melanoma/MDA-MB-435, glioblastoma/U87-MG, and triple-negative SUM159. Luciferase-tagged SUM149 and MDA-MB-435 cell lines were used to determine the incidence and the burden of locoregional and systemic spread. Mice were monitored twice a week for 9-16 weeks for percent weight loss, tumor volume and survival outcome. Metastatic tumor burden and incidence was measured as luciferase expression in lymph nodes and lungs and normalized to total protein. Results: In the SUM149 model, the saline treated control had an average tumor burden of 17.6 ± 8.6 x104 RLU per mg of protein in the ipsilateral lymph nodes (ILN). Two groups, OLM alone and CEL + OLM, had a statistically significant decrease in ILN burden. Olmesartan alone had a 7.1-fold decrease in tumor burden with an average of 2.4 ± 0.6 x104 RLU per mg of protein (p-value = 0.01 by Mann-Whitney test). Similar trend was observed for LIS, but not for HCTZ. In the subcutaneous model, synergistic antitumor activity was observed with OLM (p = 0.026) at low dose but not with LIS and CEL (p = ns). At high dose, LIS, OLM, and CEL showed significant inhibition of tumor growth but no synergy. When these agents were combined with Paclitaxel in the SUM159 model, there was only additive effect. HCTZ, an antihypertensive diuretic which has no direct impact on the vascular wall had no effect on tumor growth. Conclusion: These preclinical data strongly suggest a hitherto unappreciated role of ACE/ARB in tumor growth control and support the further exploration of fixed-dose combinations of CEL with ACE/ARB in cancer, especially inflammatory breast cancer. Citation Format: Cynthia Lee, Osmond J. D'Cruz, Kevin Ng, Vuong Trieu. Celecoxib fixed-dose combination: antitumor activity on tumor growth and metastasis [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 4902. doi:10.1158/1538-7445.AM2017-4902

Abstract 1823: Characterization of the anti-angiogenic properties of merestinib (LY2801653), an oncokinase inhibitor

Abstract Merestinib (LY2801653) is an orally bioavailable small molecule inhibitor of several oncokinases, including MET, AXL, DDR1/2, MERTK, ROS1, Tie2 (aka TEK), and MKNK1/2. Merestinib has been extensively characterized in a wide range of preclinical tumor xenograft models and shown to potently inhibit MET driven and non-MET driven tumor growth. In addition to its direct antitumor activity, merestinib inhibits angiogenesis and induces a tumor vessel normalization phenotype in xenograft tumors1. While MET signaling is important for angiogenesis, the effect of merestinib on angiogenesis is likely not exclusively driven by MET inhibition. In co-culture angiogenesis assays, merestinib inhibited VEGF-dependent and VEGF-independent endothelial cell cord formation2,3 and sprouting4 with potencies in the low nM range (3-30 nM). In contrast, the MET-specific kinase inhibitor, PF04217903, only weakly inhibited cord formation and endothelial sprouting. In an established in vivo matrigel co-implant vasculogenesis model where VEGFR2 or MET selective inhibition had minimal effect, merestinib decreased vascular density by 69%. In addition, while MET antibody emibetuzumab (human anti-MET antibody) plus ramucirumab (human anti-VEGFR2 antibody) decreased vascular density by 64%, merestinib plus ramucirumab decreased it by 92%. In a mouse adenovirus-driven VEGF-A ear angiogenesis model5, treatment with DC101, a mouse anti-VEGFR2 antibody, or merestinib inhibited angiogenesis; however the combination of DC101 and merestinib appeared to inhibit it even more. Finally, in the MKN45 gastric tumor xenograft model, merestinib (T/C = 4.8%) and DC101 (T/C = 15.3%) each significantly inhibited tumor growth alone and the combination resulted in 27.6% tumor regression and was significantly better than either single agent alone. Together, these studies indicate that merestinib has greater effects on angiogenesis than selective MET inhibition and its actions are not dependent on VEGFR2. In addition, while in vitro studies show reductions in VEGFR2 phosphorylation with high concentration of merestinib, treatment with merestinib did not inhibit VEGF dependent phosphorylation of VEGFR2 in mouse lung tissue at clinically relevant exposures. These data suggest that the anti-angiogenic activity of merestinib includes activities of other kinases targeted by merestinib. These data provide rationale and support for the clinical evaluation of combination of merestinib with ramucirumab (NCT02745769). 1-Yan et. al. Invest New Drugs. 2013;31:833-844, 2- Falcon et. al. J Hematol Oncol. 2013;6:31, 3- Falcon et. al. PLoS ONE. 2014;9:e106901, 4- Nakatsu et. al. Methods Enzymol. 2008;433:65-82, 5- Nagy et. al. Methods Enzymol. 2008;444:43-64. Citation Format: Diane M. Bodenmiller, Julie A. Stewart, Glenn F. Evans, Victoria L. Peek, Jennifer R. Stephens, Xi Lin, Seema Iyer, Beverly L. Falcon, Sudhakar Chintharlapalli, Sau-Chi Betty Yan, Anthony S. Fischl. Characterization of the anti-angiogenic properties of merestinib (LY2801653), an oncokinase inhibitor [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 1823. doi:10.1158/1538-7445.AM2017-1823

Abstract 1099: Testosterone abolishes cabazitaxel efficacy in an in vivo model of prostate cancer and affects intratumoral concentrations

Abstract Introduction Docetaxel combined with castration provides a major survival benefit in newly diagnosed metastatic PCa. Conversely, docetaxel without castration in high-risk localized PCa is not active. Cabazitaxel is a new taxane retaining its activity in tumors resistant to docetaxel and new hormonal therapies. We aimed at better characterizing in vivo the relationship between cabazitaxel and circulating androgens in a preclinical human-derived xenograft model of castration-resistant prostate cancer (CRPC). Material and methods Athymic nude mice were inoculated with the androgen receptor wt, PSA secreting CRPC cell line PC346C-DCC-K. Mice were surgically castrated when tumors were established (tumor volume (TV) of 150 mm3). After 7 days, mice were randomized to receive testosterone (40 mg) or empty pellet. The following day mice were injected with one bolus injection of cabazitaxel (33 mg/kg) or NaCl intraperitoneally. Mice were sacrificed when tumors exceeded a volume of 1,500 mm3 or the maximum follow-up of 90 days after cabazitaxel treatment. Blood was sampled biweekly for PSA and testosterone and analyzed by an immunoassay. In a second in vivo experiment intratumoral uptake of cabazitaxel was determined in the presence and absence of testosterone using a similar experimental set-up, with the exception that mice were sacrificed 7 days after cabazitaxel treatment. Here, intratumoral cabazitaxel concentrations were determined in snap frozen tumors by LC/MS-MS and corrected for tumor weight. Results Cabazitaxel treatment of castrated male mice bearing PC346C-DCC-K tumors resulted in a complete or near-complete antitumor response. In contrast, in mice supplemented with testosterone the anti-tumor effect of cabazitaxel was not significantly different from placebo (median time till TV 1,500 mm3 48 and 45 days resp.). Importantly, testosterone supplementation alone did not significantly affect tumor growth, confirming the CRPC nature of the (PC346C-DCC-K) xenograft. Interestingly, in tumors of testosterone supplemented castrated mice the intratumoral cabazitaxel concentrations were significantly decreased compared to mice that did not receive testosterone (0.39 ng cabazitaxel per mg tumor tissue vs. 1.36 ng/mg, t-test p=0.0032). Conclusions These findings indicate that circulating testosterone significantly impairs the efficacy of cabazitaxel. Testosterone supplementation may alter the metabolism of cabazitaxel, or may interfere with uptake and/or accumulation of cabazitaxel in PCa cells. We will further investigate the relationship between circulating androgens and intratumoral cabazitaxel accumulation as well as anti-tumor efficacy. The study was supported by a research grant from Sanofi Citation Format: Lisanne Mout, Corrina de Ridder, Debra Stuurman, Peter Bruijn, Ron H. Mathijssen, Ronald de Wit, Martijn P. Lolkema, Wytske M. Weerden. Testosterone abolishes cabazitaxel efficacy in an in vivo model of prostate cancer and affects intratumoral concentrations [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 1099. doi:10.1158/1538-7445.AM2017-1099

Abstract 3090: LY3207447, a tetravalent bispecific antibody targeting VEGFR2 and angiopoietin-2, provides a more efficient anti-angiogenic therapy and an alternative for combination

Abstract Angiopoietin-2 (Ang-2) is released from endothelial cells only in response to stimulus (e.g. wound healing, tumor growth) and facilitates blood vessel sprouting and inhibits pericyte-endothelial cell interaction via Tie2 signaling. In tumors, Ang-2 is up-regulated and acts together with the VEGF/VEGFR2 pathway to stimulate tumor angiogenesis and metastasis. While therapeutic intervention using antagonists to the VEGF/VEGFR2 pathway has proven to be successful in limiting disease progression in a number of different clinical settings, there is an obvious need for an improved response. In various preclinical mouse angiogenesis or xenograft models, the combination treatment with anti-Ang2 antibody and the VEGF/VEGFR blocker provided additional benefit over inhibiting the individual pathway. Here as an alternative to combo therapy, we have engineered a tetravalent IgG-scFv bispecific antibody, LY3207447. LY3207447 is an immunoglobulin G4 (IgG4) antibody, comprising of VEGFR2 antibody derived from ramucirumab and a C-terminally fused single-chain variable fragment (scFv) targeting Ang2. LY3207447 binds to both the extracellular domain of VEGFR2 and soluble Ang2 with high affinity and blocks binding of Ang2 to Tie2 and VEGF to VEGFR2, and therefore inhibits signaling. We have shown that LY3207447 blocks binding of human Ang-2 to human Tie2-Fc by an ELISA assay and neutralizes Ang-2 induced phospho-Tie-2, but not Ang-1 induced phospho-Tie-2 in CHO cells overexpressing Tie-2 receptor. Moreover, LY3207447 neutralizes human VEGF165-induced phospho-VEGFR2 stimulation, cord formation and cell proliferation in human endothelial colony forming cells (ECFCs) and human dermal microvascular endothelial cells (HMVEC-d). LY3207447 blocks human and cyno VEGFR2, but not rodent VEGFR2. For anti-Ang2 arm, LY3207447 blocks human, cyno and mouse Ang2. Pre-clinical evaluation of LY3207447 in mouse retinal angiogenesis model resulted in an abrogation of angiogenesis. Combination studies using a rodent-specific surrogate VEGFR2 blocking antibody, DC101, with parental Ang2 antibody from which the scFv was derived from, inhibited both tumor growth and metastasis, resulting in increased survival compared to monotherapies in mouse xenograft model. These data establish VEGFR2/Ang2 bispecific antibodies as a promising anti-angiogenic, anti-metastatic and anti-tumor agent for the treatment of cancer in combination with other therapies. Citation Format: Jonathan Tetreault, Sudhakar Chintharlapalli, Donmienne Leung, Damien Gerald, Linda Lee, Rowena Almonte-Baldonado, Lysiane Huber, Jianghuai Xu, Bharathi Ramamurthy, Jennifer Pereira, Johnny Croy, Jirong Lu, Ling Liu. LY3207447, a tetravalent bispecific antibody targeting VEGFR2 and angiopoietin-2, provides a more efficient anti-angiogenic therapy and an alternative for combination [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 3090. doi:10.1158/1538-7445.AM2017-3090

New HIF2α inhibitors: potential implications as therapeutics for advanced pheochromocytomas and paragangliomas.

Two recent independent studies published in Nature show robust responses of clear cell renal cell carcinoma (ccRCC) cell lines, preclinical ccRCC xenograft models and, remarkably, a patient with progressive ccRCC despite receiving multiple lines of treatment, to the long-awaited, recently developed inhibitors of hypoxia-inducible factor 2-alpha (HIF2α). This commentary published in Endocrine-Related Cancer is based on the recognition of similar molecular drivers in ccRCC and the endocrine neoplasias pheochromocytomas and paragangliomas (PPGLs), ultimately leading to stabilization of HIFs. HIF-stabilizing mutations have been detected in the von Hippel-Lindau (VHL) gene, as well as in other genes, such as succinate dehydrogenase (SDHx), fumarate hydratase (FH) and transcription elongation factor B subunit 1 (TCEB1), as well as the gene that encodes HIF2α itself: EPAS1HIF2α Importantly, the recent discovery of EPAS1 mutations in PPGLs and the results of comprehensive in vitro and in vivo studies revealing their oncogenic roles characterized a hitherto unknown direct mechanism of HIF2α activation in human cancer. The now available therapeutic opportunity to successfully inhibit HIF2α pharmacologically with PT2385 and PT2399 will certainly spearhead a series of investigations in several types of cancers, including patients with SDHB-related metastatic PPGL for whom limited therapeutic options are currently available. Future studies will determine the efficacy of these promising drugs against the hotspot EPAS1 mutations affecting HIF2α amino acids 529-532 (in PPGLs) and amino acids 533-540 (in erythrocytosis type 4), as well as against HIF2α protein activated by VHL, SDHx and FH mutations in PPGL-derived chromatin cells.

Efficient Generation of Bispecific Murine Antibodies for Pre-Clinical Investigations in Syngeneic Rodent Models

Therapeutic concepts exploiting tumor-specific antibodies are often established in pre-clinical xenograft models using immuno-deficient mice. More complex therapeutic paradigms, however, warrant the use of immuno-competent mice, that more accurately capture the relevant biology that is being exploited. These models require the use of (surrogate) mouse or rat antibodies to enable optimal interactions with murine effector molecules. Immunogenicity is furthermore decreased, allowing longer-term treatment. We recently described controlled Fab-arm exchange (cFAE) as an easy-to-use method for the generation of therapeutic human IgG1 bispecific antibodies (bsAb). To facilitate the investigation of dual-targeting concepts in immuno-competent mice, we now applied and optimized our method for the generation of murine bsAbs. We show that the optimized combinations of matched point-mutations enabled efficient generation of murine bsAbs for all subclasses studied (mouse IgG1, IgG2a and IgG2b; rat IgG1, IgG2a, IgG2b, and IgG2c). The mutations did not adversely affect the inherent effector functions or pharmacokinetic properties of the corresponding subclasses. Thus, cFAE can be used to efficiently generate (surrogate) mouse or rat bsAbs for pre-clinical evaluation in immuno-competent rodents.

DS-8201a, a HER2-targeting antibody-drug conjugate, to elicit immune responses and benefits in combination with an anti-PD-1 antibody.

1031 Background: DS-8201a, a HER2-targeting antibody–drug conjugate (ADC), with a topoisomerase I inhibitor, exatecan drivative (DX-8951 derivative, DXd) has been shown to have antitumor effects in preclinical xenograft models and clinical trials, but the involvement of the immune system in the antitumor efficacy of DS-8201a has not been elucidated yet. Methods: The antitumor efficacy of DS-8201a individually and in combination with an anti-PD-1 antibody was determined in a syngeneic mouse model with human HER2-expressing CT26.WT (CT26.WT-hHER2) cells. Mice whose tumors had been cured by DS-8201a treatment were rechallenged with CT26.WT-hHER2 cells; their splenocytes were co-cultured with CT26.WT-hHER2 or CT26.WT-mock cells, and IFN-g secretion by these cells was determined. To investigate effects of DXd and DS-8201a on dendritic cells (DCs), the expression of DC markers on bone marrow derived DCs (BMDCs) and intratumoral DCs was analyzed by flow cytometry. Furthermore, MHC class I and PD-L1 expression on tumor cells was analyzed. Results: At a weekly dosage of 10 mg/kg, DS-8201a showed significant antitumor effects in the mouse model. Mice whose tumors had been cured by DS-8201a treatment rejected the rechallenge with CT26.WT-hHER2 cells, and splenocytes from these mice were activated by both CT26.WT-hHER2 and CT26.WT-mock cells. In the mouse model, DS-8201a treatment raised a population of intratumoral DCs (CD45+CD11c+MHC class II+) and increased DC expression of CD86, a DC activation marker; DXd also up-regulated CD86 expression on BMDCs in vitro. Furthermore, DS-8201a up-regulated PD-L1 and MHC class I expression on tumor cells. Notably, antitumor effects of the combination of DS-8201a with an anti-PD-1 antibody were better than those of monotherapy. Conclusions: DS-8201a elicits immune responses via mechanisms other than cytotoxic effects on tumor cells. This finding suggests additional benefits of combining DS-8201a with an immune checkpoint inhibitor (ICI). The combination of DS-8201a and an anti-PD-1 antibody was effective in tumor suppression, indicating that DS-8201a may be successfully combined with an ICI in human clinical applications.

Antitumor activity of indenoisoquinoline inhibitors of topoisomerase 1 (TOP1) via apoptosis and autophagocytosis pathways in animal models.

11588 Background: We performed pharmacodynamic biomarker analysis for response to a panel of three indenoisoquinolines (LMP776, LMP400 and NSC706744, J. Med. Chem. 49:7740, 2006) that have demonstrated anti-tumor activity in dogs. In preclinical xenograft models treated with indenoisoquinolines, we observed that gH2AX was not a useful biomarker for the biological activity of compound 706744, but was a reasonable biomarker of drug activity (Clin. Canc. Res. 16:5447, 2010) for the other two compounds, even though in vitro data indicated that all 3 compounds inhibited TOP1 and killed tumor cells. It has been reported that irinotecan activates autophagy (Pharm. Rev. 65:1162, 2013) which correlated with its metabolism to SN-38; we therefore developed, validated and tested an immunofluorescence microscopy assay for LC3 as a marker of autophagy. Methods: Assays were developed to evaluate apoptosis by co-localization of cleaved caspase 3 and gH2Ax, and autophagy by LC3 immunofluorescence on formalin fixed, paraffin-embedded tissue sections of xenografts models or lymphomas from outbred dogs. Percent positive cells containing LC3 puncta were quantitated using a spot morphology algorithm. Analysis of gH2AX and cleaved caspase 3 cellular co-localization was developed using a blebbing morphology algorithm (Definiens). Results: LC3 reported that indenoisoquinoline 706744 activates autophagy in vitro with the absence of cleaved caspase 3-dependent apoptosis while the -776 and -400 compounds do not activate autophagy, but instead demonstrate apoptosis in response to drug treatment. Results in animal models confirmed that both autophagy and apoptosis were active. Clinical readiness of the assays was confirmed on canine biopsy FFPE slides. Conclusions: 1,Structurally-related TOP1 inhibitors may trigger alternative pathways of cell destruction; 2,Autophagy may report drug anti-tumor activity or tumor drug resistance according to current literature. This assay may be useful for determination of pharmacodynamic pathways associated with anti-tumor activity to elucidate mechanism of action of investigational agents used in clinical trials.

Acetazolamide potentiates the anti-tumor potential of HDACi, MS-275, in neuroblastoma

BackgroundNeuroblastoma (NB), a tumor of the primitive neural crest, despite aggressive treatment portends a poor long-term survival for patients with advanced high stage NB. New treatment strategies are required.MethodsWe investigated coordinated targeting of essential homeostatic regulatory factors involved in cancer progression, histone deacetylases (HDACs) and carbonic anhydrases (CAs).ResultsWe evaluated the antitumor potential of the HDAC inhibitor (HDACi), pyridylmethyl-N-{4-[(2-aminophenyl)-carbamoyl]-benzyl}-carbamate (MS-275) in combination with a pan CA inhibitor, acetazolamide (AZ) on NB SH-SY5Y, SK-N-SH and SK-N-BE(2) cells. The key observation was that the combination AZ + MS-275 significantly inhibited growth, induced cell cycle arrest and apoptosis, and reduced migration capacity of NB cell line SH-SY5Y. In addition, this combination significantly inhibited tumor growth in vivo, in a pre-clinical xenograft model. Evidence was obtained for a marked reduction in tumorigenicity and in the expression of mitotic, proliferative, HIF-1α and CAIX. NB xenografts of SH-SY5Y showed a significant increase in apoptosis.ConclusionMS-275 alone at nanomolar concentrations significantly reduced the putative cancer stem cell (CSC) fraction of NB cell lines, SH-SY5Y and SK-N-BE(2), in reference to NT2/D1, a teratocarcinoma cell line, exhibiting a strong stem cell like phenotype in vitro. Whereas stemness genes (OCT4, SOX2 and Nanog) were found to be significantly downregulated after MS-275 treatment, this was further enhanced by AZ co-treatment. The significant reduction in initial tumorigenicity and subsequent abrogation upon serial xenografting suggests potential elimination of the NB CSC fraction. The significant potentiation of MS-275 by AZ is a promising therapeutic approach and one amenable for administration to patients given their current clinical utility.

Abstract P6-12-03: A phase I study of AZD9496, a novel oral, selective estrogen receptor degrader (SERD) in women with estrogen receptor positive, HER-2 negative advanced breast cancer (ABC)

Abstract Background AZD9496 is a potent orally bioavailable ER antagonist and degrader that has shown antitumor efficacy in a range of preclinical xenograft models including ESR1wild-type tamoxifen-resistant and long term estrogen deprived models and an ESR1 mutant model. Methods This is a phase I, open label global multicenter study in women with ER+ HER2–ve BC either metastatic or locoregionally recurrent, not amenable to treatment with curative intent. Patients are post-menopausal, or pre-menopausal women receiving LHRH agonist therapy, with disease progression after ≥6 months endocrine therapy for ER+ BC (no limit on number of prior endocrine therapies; ≤2 prior chemotherapies in advanced setting). The primary objective is to determine the safety and tolerability of AZD9496. Cohorts of 3-6 patients received daily oral therapy and dose limiting toxicities (DLTs) occurring in cycle 1 (28 days) were assessed. Patients are dosed until MTD (defined as ≤1/6 patients with a DLT) or maximum feasible dose (MFD) is reached. Key secondary objectives include determination of single and multiple dose pharmacokinetics (PK), and preliminary antitumor efficacy. ER target modulation by protein and gene expression is evaluated in circulating tumor cells and paired tumor biopsies. In addition to the dose escalation phase, expansion cohort(s) in patients with or without ESR1 mutations can be enrolled to examine the safety, tolerability, PK and biological activity of AZD9496 further. Results Preliminary data as of 30th April 2016: 45 patients (median age 62 (range 41-83); 38 post-menopausal, 7 pre/perimenopausal; visceral metastases 76%, prior fulvestrant 25/45) received AZD9496 in 7 dose escalation cohorts: 20mg QD n=4, 40mg BID n=6, 80mg BID n=5, 150mg BID n=6, 250mg BID n=6, 400mg BID n=6, 600mg BID n=6 and also a 250mg BID expansion cohort n=6. The majority of adverse events (AEs) were grade 1 or 2; the most common treatment-related AEs (≥10%) have been diarrhoea (33%), fatigue (27%), nausea (22%), upper abdominal pain (13%) and increased liver function tests (13%). Six patients had treatment-related grade 3 AEs, 5 of which were manageable with dose interruption +/- dose reduction. Specifically, three had DLTs: grade 3 increased AST/ALT/GGT-150mg BID, serious adverse reaction (SAR) leading to withdrawal; grade 3 diarrhoea and grade 3 increased AST/ALT/GGT-400mg BID, SAR, manageable with dose reductions; grade 3 diarrhoea 600mg BID, manageable with dose reduction. The MTD/MFD has not been reached. Following the first dose up to 400mg the AZD9496 exposure increased in reasonable proportion to increasing dose. At 600mg a more than dose-proportional increase in exposure was observed. Evidence of reduced ER and Ki67 has been observed in on-study biopsies at 150mg BID and above. 10 subjects received treatment for >3-<6 months (5 ongoing, 5 discontinued),4 subjects >6-<12 months (3 ongoing, 1 discontinued), 3 subjects ≥1 year (2 ongoing, 1 discontinued). Conclusions AZD9496 has a tolerable safety profile, evidence of PD biomarker modulation and prolonged stabilisation of disease in women with heavily pre-treated ER+ve ABC. Citation Format: Hamilton E, Patel M, Armstrong A, Baird R, Jhaveri K, Hoch M, Morgan S, Dowdall T, Schiavon G, Klinowska T, Weir H, Bujac S, Nash T, Im S-A. A phase I study of AZD9496, a novel oral, selective estrogen receptor degrader (SERD) in women with estrogen receptor positive, HER-2 negative advanced breast cancer (ABC) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-12-03.

NF-\u03baB suppresses apoptosis and promotes bladder cancer cell proliferation by upregulating survivin expression in vitro and in vivo

Nuclear factor kappa-B (NF-κB) activation is a common phenomenon in cancers, which results in the aberrant expression of NF-κB target genes and leads to malignant transformation, metastatic dissemination, abnormal cell proliferation or resistance to cell death. Survivin is a unique member of the IAP family, a well-known cancer-specific molecule and a molecular marker of poor clinical outcome in several cancer types, including bladder cancer. YM-155, a potent survivin suppressor, has been shown to have anti-tumor activity in preclinical cell lines, xenograft models and phase I/II studies. In the present study, we investigated the function of the NF-κB/survivin pathway in bladder cancer. We found that NF-κB can promote cell cycle progression and reduce apoptosis by upregulating survivin expression, thereby increasing cellular proliferation. We further confirmed the tumorigenic function of the NF-κB/survivin pathway in vivo using a xenograft tumor model of stable NF-κB-overexpressing 5637 cells. Moreover, we found that YM-155 significantly induced apoptosis and decreased cellular proliferation as well as tumor growth in mice. Our results demonstrate the carcinogenic function of the NF-κB/survivin pathway in bladder cancer and the role of YM-155 as a promising agent for the strategic treatment of bladder cancer.

Discovery and Preclinical Development of Novel CD74-Targeting Antibody-Drug Conjugates (ADCs) with Significant Activity in Multiple Myeloma (MM) Cell Lines and Xenograft Models

CD74, also known as HLA-DR-associated invariant chain, is a type II transmembrane glycoprotein highly expressed in many B-cell malignancies. The limited expression of CD74 in normal tissues suggests it may be a suitable ADC target for these tumor types. Accordingly, we engineered an anti-CD74 human IgG1 antibody (SP7219) using novel Fab-based ribosome display methods. The selected Fabs were readily reformatted and directly screened as IgGs using Sutro’s unique high-throughput, cell-free protein synthesis platform, Xpress CFTM. We then developed novel, potent ADCs, SP7676 and SP7675 (STRO-001), comprised of our lead antibody (SP7219) conjugated to non-cleavable DBCO-maytansinoid linker-warheads with an average drug-antibody ratios (DAR) of 2. We used site-specific conjugation technology which results in a high degree of homogeneity characterized by the drug linker covalently binding to a single defined site. The sites for conjugation were selected based on highest cell killing activity and stablity in vitro and in vivo. Both ADCs demonstrate potent cell killing activity across multiple B-cell tumor lines in vitro, and anti-tumor activity in preclinical multiple myeloma xenograft models. In vitro cytotoxicity assays show nanomolar potency of STRO-001 in four MM cell lines: Mc/CAR (IC50 0.8 nM), MM.1S (IC50 10-11 nM), U266B1 (IC50 8.5 -9.3 nM), and ARP-1 (IC50 4.3-22 nM). CD74 cell surface expression is required for ADC anti-proliferative effect but the expression level does not seem to correlate with in vitro potency. SP7676 elicited a robust anti-tumor response in the ANBL-6 multiple myeloma xenograft model. Durable regressions were observed in all mice at ≥ 3 mg/kg, with equivalent efficacy (regression) at 3 mg/kg (every 3 days x5) and 10 mg/kg (every 3 days x5 or weekly x3). SP7676 also elicited a clear survival benefit in a disseminated multiple myeloma CAG xenograft model starting at 1mg/kg every 3 days x 5 doses. Similarly, both SP7676 and STRO-001 inhibited the formation of internal visceral tumors in the ARP-1 xenograft model after 3 weekly doses of 3 mg/kg. Evaluation of our lead candidate, STRO-001 in additional MM cell lines and primary patient samples is planned. The tolerability of STRO-001 in non-human primates is under evaluation. STRO-001 was administered to cynomolgous monkeys in an exploratory dose-escalating study up to 30 mg/kg x 2 doses on Day 1 and 15. STRO-001 reduces normal B-cell populations at ≥1 mg/kg after a single dose, providing pharmacodynamic evidence of B-cell targeting while other hematopoietic lineages are mostly affected only at the highest dose studied. Anticipated hematologic toxicities were readily reversible at 1, 3 and 10 mg/kg and target organs of interest were identified. Based on these encouraging data, STRO-001 is advancing to IND-enabling studies for the treatment of CD74 expressing multiple myeloma and other B-cell malignancies. DisclosuresAbrahams:Sutro Biopharma: Employment. Li:Sutro Biopharma: Employment. Yu:Sutro Biopharma: Employment. Krimm:Sutro Biopharma: Employment. Kahana:Celgene: Employment. Narla:Celgene: Employment. Schwartz:Celgene: Employment. Boylan:Celgene: Employment. Hoffmann:Sutro Biopharma: Employment. Steiner:Sutro Biopharma: Employment. Zawada:Sutro Biopharma: Employment. Stephenson:Sutro Biopharma: Employment. Bruhns:Sutro Biopharma: Employment. DeAlmeida:Sutro Biopharma: Employment. Matheny:Sutro Biopharma: Employment. Bussell:Sutro Biopharma: Employment. Galan:Sutro Biopharma: Employment. Kline:Sutro Biopharma: Employment. Vasquez:Sutro Biopharma: Employment. Yam:Sutro Biopharma: Employment. Stafford:Sutro Biopharma: Employment. Heinsohn:Sutro Biopharma: Employment. Sato:Sutro Biopharma: Employment. Molina:Sutro Biopharma: Employment. Hallam:Sutro Biopharma: Employment. Lupher:Sutro Biopharma: Employment.

CD47 Monoclonal Antibody SRF231 Is a Potent Inducer of Macrophage-Mediated Tumor Cell Phagocytosis and Reduces Tumor Burden in Murine Models of Hematologic Malignancies

The transmembrane protein CD47 is an immunoglobulin superfamily member involved in multiple cellular processes, including cell migration, adhesion and T cell function. The interaction between CD47 and signal regulatory protein alpha (SIRPα), an inhibitory protein expressed on macrophages, prevents phagocytosis of CD47-expressing cells. CD47 was originally identified as a tumor antigen on human ovarian cancer and was subsequently shown to be expressed on multiple human tumor types, including both hematologic and solid tumors. In many hematologic cancers, high CD47 expression is associated with poor clinical outcomes. CD47 is also expressed at low levels on virtually all non-malignant cells, and loss of expression or changes in membrane distribution may serve as markers of aged or damaged cells, particularly on red blood cells (RBC). High expression of CD47 on tumors blocks phagocytic uptake, subsequent antigen cross-presentation and T cell activation, which collectively contribute to tumor immune evasion. Agents that block the CD47-SIRPα interaction can restore phagocytic uptake of CD47+ target cells and lower the threshold for macrophage activation, which can enhance the efficacy of therapeutic antibodies with ADCC-enabling activity. We developed and characterized a CD47 blocking antibody and evaluated its activity in multiple hematologic models.SRF231 is a fully human monoclonal antibody that binds with high affinity to human CD47 and blocks the CD47-SIRP□ interaction. SRF231 promotes macrophage-mediated phagocytic clearance of several hematologic primary tumor samples and cell lines in vitro. For example, SRF231 increases phagocytosis of Raji tumor cell line targets with an EC50 of ~300 ng/ml. Enhanced phagocytosis is preferential for tumor cells over normal leukocytes and RBC. Tumor cell phagocytosis can be enhanced in the presence of opsonizing antibodies (e.g., anti-CD20 Ab) when co-administered with SRF231.In vivo efficacy of SRF231 was assessed in several preclinical murine xenograft models of hematologic malignancies. Notably, SRF231 administration led to profound tumor growth inhibition in models of multiple myeloma, diffuse large B cell lymphoma and Burkitt's lymphoma as a single agent and in combination with opsonizing antibodies. In the Raji xenograft model, single agent therapy leads to 112% tumor growth inhibition. This anti-tumor activity is at least partially dependent on macrophages, as depletion of macrophages via clodronate administration leads to reduced tumor growth inhibition. Tumor-associated macrophage (TAM) numbers and polarization status are also modulated by SRF231 treatment.In summary, the CD47 mAb SRF231 induces robust tumor cell phagocytosis and tumor clearance both alone and in combination with opsonizing antibodies in pre-clinical models of myeloma and lymphoma. These properties warrant further development of SRF231 in hematologic malignancies. SRF231 is currently in IND-enabling studies and is expected to enter clinical trials in 2017. DisclosuresNo relevant conflicts of interest to declare.

Dual targeting of glutaminase 1 and thymidylate synthase elicits death synergistically in NSCLC.

Glutaminase 1 (GLS1) expression is increased in non-small cell lung cancer (NSCLC). GLS1 knockdown using siRNA or inhibition using bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) induced cell cycle arrest with significant reduction of ATP level while levels of reactive oxygen species or glutathione were not affected in NSCLC cell lines. Recently we found that NSCLC significantly depends on cytosol NADH for ATP production. GLS1 remarkably contributes to ATP production through transferring cytosolic NADH into mitochondria via malate-aspartate shuttle by supply of glutamate in NSCLC. Regulation of malate-aspartate shuttle by knockdown or inhibition of glutamic-oxaloacetic transaminase 2 or malate dehydrogenase 2 mimicked GLS1 knockdown, which induced cell death with ATP reduction in NSCLC. Therefore, GLS1 inhibition induced cell cycle arrest with ATP depletion by glutamate reduction. Dual inhibition with BPTES and thymidylate synthase inhibitor, 5-fluorouracil (5-FU), elicits cell death synergistically through cell cycle arrest in NSCLC. A preclinical xenograft model of NSCLC showed remarkable anti-tumour effect synergistically in the BPTES and 5-FU dual therapy group.

Peginterferon Beta-1a Shows Antitumor Activity as a Single Agent and Enhances Efficacy of Standard of Care Cancer Therapeutics in Human Melanoma, Breast, Renal, and Colon Xenograft Models.

Because of its tumor-suppressive effect, interferon-based therapy has been used for the treatment of melanoma. However, limited data are available regarding the antitumor effects of pegylated interferons, either alone or in combination with approved anticancer drugs. We report that treatment of human WM-266-4 melanoma cells with peginterferon beta-1a induced apoptotic markers. Additionally, peginterferon beta-1a significantly inhibited the growth of human SK-MEL-1, A-375, and WM-266-4 melanoma xenografts established in immunocompromised mice. Peginterferon beta-1a regressed large, established WM-266-4 xenografts in nude mice. Treatment of SK-MEL-1 tumor-bearing mice with a combination of peginterferon beta-1a and the MEK inhibitor PD325901 ((R)-N-(2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodophenylamino)benzamide) significantly improved tumor growth inhibition compared with either agent alone. Examination of the antitumor activity of peginterferon beta-1a in combination with approved anticancer drugs in breast and renal carcinomas revealed improved antitumor activity in these preclinical xenograft models, as did the combination of peginterferon beta-1a and bevacizumab in a colon carcinoma xenograft model.

Tumor Xenografts of Human Clear Cell Renal Cell Carcinoma But Not Corresponding Cell Lines Recapitulate Clinical Response to Sunitinib: Feasibility of Using Biopsy Samples

BackgroundParallel development of preclinical models that recapitulate treatment response observed in patients is central to the advancement of personalized medicine. ObjectiveTo evaluate the use of biopsy specimens to develop patient-derived xenografts and the use of corresponding cell lines from renal cell carcinoma (RCC) tumors for the assessment of histopathology, genomics, and treatment response. Design, setting, and participantsA total of 74 tumor specimens from 66 patients with RCC were implanted into immunocompromised NOD-SCID IL2Rg−/− mice. Four cell lines generated from patients’ specimens with clear cell pathology were used for comparative studies. Outcome measurements and statistical analysisPreclinical models were established and assessed. Engraftment rates were analyzed using chi-square testing. Analysis of variance (two-way analysis of variance) was conducted to assess tumor growth. Results and limitationsOverall, 33 RCC mouse xenograft models were generated with an overall engraftment rate of 45% (33 of 74). Tumor biopsies engrafted comparably with surgically resected tumors (58% vs 41%; p=0.3). Xenograft tumors and their original tumors showed high fidelity in regard to histology, mutation status, copy number change, and targeted therapy response. Engraftment rates from metastatic tumors were higher but not more significant than primary tumors (54% vs 34%; p=0.091). Our engraftment rate using metastases or biopsies was comparable with recent reports using resected primary tumors. In stark contrast to corresponding cell lines, all tested xenografts recapitulated patients’ clinical response to sunitinib. ConclusionsPatient-derived xenograft models can be effectively established from tumor biopsies. Preclinical xenograft models but not matched cell lines reflected clinical responses to sunitinib. Patient summaryMatched patient-derived clear cell renal cell carcinoma xenografts and cell lines from responsive and refractory patients treated with sunitinib were established and evaluated for pharmacologic response to anti–vascular endothelial growth factor treatment. Both models accurately reflected the genetic characteristics of original tumors, but only xenografts recapitulated drug responses observed in patients. These models could serve as a powerful platform for precision medicine.

Induction of ferroptotic cell death for overcoming cisplatin resistance of head and neck cancer

Inhibition of key molecules related to ferroptosis, cystine/glutamate antiporter and glutathione peroxidase, may induce eradication of chemotherapy/radiotherapy-resistant cancer cells. The present study investigated whether ferroptosis could overcome head and neck cancer (HNC) resistance to cisplatin treatment. Three cisplatin-resistant HNC cell lines (AMC-HN3R, -HN4R, and -HN9R) and their parental lines were used. The effects of cystine and glutamate alteration and pharmacological and genetic inhibition of cystine/glutamate antiporter were assessed by measuring viability, death, reactive oxygen species production, protein expression, and preclinical mouse tumor xenograft models. Conditioned media with no cystine or glutamine excess induced ferroptosis of both cisplatin-sensitive and -resistant HNC cells without any apparent changes to necrosis and apoptosis markers. The cystine/glutamate antiporter inhibitors erastin and sulfasalazine inhibited HNC cell growth and accumulated lipid reactive oxygen species, thereby inducing ferroptosis. Genetic silencing of cystine/glutamate antiporter with siRNA or shRNA treatment also induced effective ferroptotic cell death of resistant HNC cells and enhanced the cisplatin cytotoxicity of resistant HNC cells. Pharmacological and genetic inhibition of cystine/glutamate antiporter significantly sensitized resistant HNC cells to cisplatin in vitro and in vivo. Pharmacological and genetic inhibition of cystine/glutamate antiporter overcomes the cisplatin resistance of HNC cells by inducing ferroptosis.

Abstract 1285: Tumor associated macrophages can process antibody-drug conjugates and contribute to antitumor activity in preclinical xenograft models

Abstract The primary mechanism of antibody-drug conjugates (ADCs) is the targeted delivery of a cytotoxic payload via cancer antigen mediated internalization. However, stromal components in the tumor microenvironment may also play a role in ADC penetration, distribution, and processing. Here, we study the potential roles of Fc-FcγR interaction between tumor-associated macrophages (TAMs) and ADCs in the antitumor activity observed in xenograft models. In the CD30+ L428 Hodgkin lymphoma (HL) model, the anti-CD30 ADC (cAC10-vcMMAE) and a non-binding control ADC (h00-vcMMAE) showed similar antitumor activity over a two-week period post treatment initiation. The antitumor activity was correlated with payload release, as h00-vcMMAE produced intratumoral MMAE concentration comparable to cAC10-vcMMAE in this time frame. Histopathology analysis of L-428 xenografts revealed high abundance of TAMs, leading to the hypothesis that TAMs can internalize and process ADCs for payload release. Using immunohistochemistry and flow cytometry, we confirmed that h00-vcMMAE bound to macrophage cell lines and TAMs. We further examined the presence of TAMs in additional xenograft models and correlated that to the antitumor activity of non-binding h00-vcMMAE. High levels of TAMs were observed in the KM-H2 HL and BR620 breast cancer models that were sensitive to h00-vcMMAE. In contrast, xenograft models with much fewer TAMs (DOHH2, SU-DHL8, and Karpas-299) did not respond to h00-vcMMAE treatment. Interestingly, h00-vcMMAF, releasing a membrane non-permeable payload, had no activity on the L-428 tumor model. These data suggest TAM-processed drug can mediate bystander tumor killing when the released payload is membrane permeable. To evaluate whether Fc-FcγR interaction plays a role in the ADC uptake by TAMs, we mutated the Fc region of h00-vcMMAE to decrease FcγR binding affinity (E233P:L234V:L235, G1V1). h00G1V1-vcMMAE lost its cytotoxicity activity in FcγR+ THP-1 monocytes. Furthermore, h00G1V1-vcMMAE could no longer mediate tumor regression or growth delay in three xenograft models that are sensitive to h00-vcMMAE treatment. These results suggest ADC-FcγR interaction is required for ADC processing by TAMs in these xenografts. These results suggest that TAMs can contribute to ADC processing through FcγR interaction in preclinical tumor models. Moreover, TAM-processed membrane permeable payload can mediate bystander tumor cell killing and contribute to ADC activity. Although this phenomenon may be an additional mechanism of ADCs in vivo, whether TAMs play a role in patients’ response to ADCs requires further correlated studies in clinical trials. Citation Format: Fu Li, Michelle Ulrich, Mechthild Jonas, Germein Linares, Xinqun Zhang, Lori Westendorf, Dennis Benjamin, Che-Leung Law. Tumor associated macrophages can process antibody-drug conjugates and contribute to antitumor activity in preclinical xenograft 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 1285.

Abstract 4623: Reevaluation of the role of Notch signaling in melanoma tumor development, melanoma cell survival and drug resistance

Abstract Notch is one of the simplest signaling pathways that is required for almost every cell type in the body. It plays several roles such as cell fate decision, differentiation and stem cell maintenance. Notch signaling plays a role in the maintenance of melanoblasts and melanocyte stem cells of the epidermis by preventing their apoptosis. Notch signaling has also been implicated in different aspects of melanoma biology including tumor initiation, progression and metastasis. Accordingly, Notch has been targeted for melanoma therapy both in preclinical xenograft models as well as clinical trials. Notch inhibitors, mainly γ-secretase inhibitors, have been widely used for this purpose. However, clinical trials with these agents have not been proven to be highly effective for melanoma treatment. In previously published studies we showed that Notch signaling is involved in regulation of melanoma trans-differentiation, a prognostic feature in primary melanoma. In this study, we reevaluated the role of Notch in melanoma development, cell survival and sensitivity to BRAF and MEK inhibitors. First, our preliminary data using the BRAFV600E/Pten mouse model showed that topical application of γ-secretase inhibitor DAPT delays tumor formation and growth. In several early passage metastatic human melanoma cell lines, we found that Notch receptors (Notch 1-3) are highly upregulated compared to normal epidermal melanocytes. Interestingly, mRNA levels for Notch1-3 receptors in normal melanocytes were either higher or comparable to their levels in the cell lines. This suggested that Notch receptor levels are tightly regulated in normal melanocytes by post-transcriptional mechanisms. Presence of cleaved Notch intracellular domain (NICD), both cytoplasmic and nuclear, in melanoma cells, but not in melanocytes, indicated constitutive activation of Notch signaling in melanoma cells. However, inhibition of Notch activation by treatment with γ-secretase inhibitor had no significant effect on survival of melanoma cells in vitro. Thus, Notch signaling appears to be dispensable for melanoma cell survival. Additionally, inhibition of Notch activation did not alter the sensitivity of melanoma cells to BRAF(V600E) and MEK inhibitors. Our results highlight the need for re-examination of Notch-regulated melanoma trans-differentiation as a therapeutic outcome. Citation Format: Dareen Mikheil, Carlos Rodriguez, Ashika Jayanthi, Prithvi Yarlagadda, Vijaysaradhi Setaluri. Reevaluation of the role of Notch signaling in melanoma tumor development, melanoma cell survival and drug resistance. [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 4623.

Abstract 3493: New, highly potent antibodies to death receptors having Fc mutations to increase antitumor activity

Abstract Death receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2 or Apo2) are TNF receptor superfamily members that are receptors for TRAIL (Apo2 ligand), an immunomodulatory cytokine. Binding of TRAIL to DR4 or DR5 can activate the extrinsic apoptotic pathway selectively in tumor cells. Agonist antibodies to death receptors DR4 and DR5 thus have potential for treatment of cancer and may be better than derivatives of TRAIL itself because of their superior pharmacokinetics and receptor selectivity. However, despite dramatic effects in vitro and in preclinical xenograft models, several agonist antibodies to DR4 and especially DR5 have not provided significant therapeutic benefit in clinical trials. While resistance mechanisms in cancer cells are undoubtedly one reason, another factor may be inadequate potency of the antibodies tested. We therefore used intense immunization and screening protocols to develop very potent anti-DR4 and anti-DR5 monoclonal antibodies (mAbs), which were humanized to make the mAbs denoted HuD114 and HuG4.2 respectively. These mAbs were substantially more effective at killing tumor cells in vitro than the mAbs that have been tested in clinical trials. In addition, we introduced one or two mutations into the Fc (constant) regions of these and other potent anti-DR4 and anti-DR5 mAbs in order to increase affinity for the Fc gamma receptor IIb. Cross-linking, as provided by binding of anti-DR4 and anti-DR5 mAbs to Fc gamma receptors on immune cells, is required for effective transmission of an apoptotic signal through the death receptors. Accordingly, introduction of these mutations greatly increased the ability of the mAbs to kill tumor cells in vitro in the presence of human peripheral blood mononuclear cells, and to inhibit the growth of tumor xenografts in mouse models, with two mutations generally more effective than a single mutation. As a complementary approach to increase cross-linking, we also developed bispecific antibodies containing two binding domains from an anti-DR4 mAb and two binding domains from an anti-DR5 mAb. These bispecific mAbs were made in the IgG-like Bs(scFv)4-IgG format consisting of a homodimer of two monomers, each monomer having a single chain Fv from an anti-DR4 mAb linked to a heavy chain constant region, and a single chain Fv from an anti-DR5 mAb linked to a light chain constant region. Such a bispecific mAb was more effective at killing tumor cells than an anti-DR4 or anti-DR5 mAb alone, or even a mixture of anti-DR4 and anti-DR5 mAbs. Based on their potency in vitro and in animal models, we believe that HuD114 and HuG4.2, enhanced by the Fc mutations and/or in bispecific form, have the potential for greater clinical efficacy than previously tested anti-DR4 and anti-DR5 mAbs and thus merit further investigation. Citation Format: Lihong Wang, Yi Ding, Hangil Park, April Zhang, Zhengran Wang, Maximiliano Vasquez, Cary Queen, Jin Kim. New, highly potent antibodies to death receptors having Fc mutations to increase antitumor activity. [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 3493.