Cell Lung Cancer Xenograft Model Research Articles

Magnolol Induces Apoptosis and Suppresses Immune Evasion in Non-small Cell Lung Cancer Xenograft Models.

Non-small cell lung cancer is known for its rapid growth and immune evasion, demanding effective therapies targeting both tumor cells and the microenvironment. Magnolol has shown promising anti-tumor effects in various cancers. CL1-5-F4-bearing mice were divided into control, 40 mg/kg, and 60 mg/kg magnolol groups, once tumors reached 100 mm3 Tumor growth and body weight were monitored biweekly, and after 13 days, mice were euthanized for tumor and organ collection for subsequent staining. Histopathology and serum biochemistry assessed organ toxicity. Magnolol dose-dependently suppressed NSCLC progression, with no pathology alterations observed in normal organs. Magnolol-induced apoptosis and cell cycle arrest, evidenced by increased cleaved caspase-3 and decreased cyclin D1/CDK4 levels. It also down-regulated VEGF, FOXP3, and IDO-1 in tumors, implicating tumor microenvironment modulation. Magnolol exhibits significant antitumor effects in NSCLC by inducing apoptosis, inhibiting proliferation, and modulating the tumor microenvironment. These results support further investigation of magnolol as a therapeutic adjuvant to enhance NSCLC treatment outcomes.

The SOS1 Inhibitor MRTX0902 Blocks KRAS Activation and Demonstrates Antitumor Activity in Cancers Dependent on KRAS Nucleotide Loading.

KRAS is the most frequently mutated oncogene in human cancer and facilitates uncontrolled growth through hyperactivation of the receptor tyrosine kinase (RTK)/mitogen-activated protein kinase (MAPK) pathway. The Son of Sevenless homolog 1 (SOS1) protein functions as a guanine nucleotide exchange factor (GEF) for the RAS subfamily of small GTPases and represents a druggable target in the pathway. Using a structure-based drug discovery approach, MRTX0902 was identified as a selective and potent SOS1 inhibitor that disrupts the KRAS:SOS1 protein-protein interaction to prevent SOS1-mediated nucleotide exchange on KRAS and translates into an anti-proliferative effect in cancer cell lines with genetic alterations of the KRAS-MAPK pathway. MRTX0902 augmented the antitumor activity of the KRAS G12C inhibitor adagrasib when dosed in combination in eight out of 12 KRAS G12C-mutant human non-small cell lung cancer and colorectal cancer xenograft models. Pharmacogenomic profiling in preclinical models identified cell cycle genes and the SOS2 homolog as genetic co-dependencies and implicated tumor suppressor genes (NF1 and PTEN) in resistance following combination treatment. Lastly, combined vertical inhibition of RTK/MAPK pathway signaling by MRTX0902 with inhibitors of EGFR or RAF/MEK led to greater downregulation of pathway signaling and improved antitumor responses in KRAS-MAPK pathway-mutant models. These studies demonstrate the potential clinical application of dual inhibition of SOS1 and KRAS G12C and additional SOS1 combination strategies that will aide in the understanding of SOS1 and RTK/MAPK biology in targeted cancer therapy.

DB-1310, an ADC comprised of a novel anti-HER3 antibody conjugated to a DNA topoisomerase I inhibitor, is highly effective for the treatment of HER3-positive solid tumors

BackgroundHER3 (ErbB3), a member of the human epidermal growth factor receptor family, is frequently overexpressed in various cancers. Multiple HER3-targeting antibodies and antibody–drug conjugates (ADCs) were developed for the solid tumor treatment, however none of HER3-targeting agent has been approved for tumor therapy yet. We developed DB-1310, a HER3 ADC composed of a novel humanized anti-HER3 monoclonal antibody covalently linked to a proprietary DNA topoisomerase I inhibitor payload (P1021), and evaluate the efficacy and safety of DB-1310 in preclinical models.MethodsThe binding of DB-1310 to Her3 and other HER families were measured by ELISA and SPR. The competition of binding epitope for DB-1310 and patritumab was tested by FACS. The sensitivity of breast, lung, prostate and colon cancer cell lines to DB-1310 was evaluated by in vitro cell killing assay. In vivo growth inhibition study evaluated the sensitivity of DB-1310 to Her3 + breast, lung, colon and prostate cancer xenograft models. The safety profile was also measured in cynomolgus monkey.ResultsDB-1310 binds HER3 via a novel epitope with high affinity and internalization capacity. In vitro, DB-1310 exhibited cytotoxicity in numerous HER3 + breast, lung, prostate and colon cancer cell lines. In vivo studies in HER3 + HCC1569 breast cancer, NCI-H441 lung cancer and Colo205 colon cancer xenograft models showed DB-1310 to have dose-dependent tumoricidal activity. Tumor suppression was also observed in HER3 + non-small cell lung cancer (NSCLC) and prostate cancer patient-derived xenograft (PDX) models. Moreover, DB-1310 showed stronger tumor growth-inhibitory activity than patritumab deruxtecan (HER3-DXd), which is another HER3 ADC in clinical development at the same dose. The tumor-suppressive activity of DB-1310 synergized with that of EGFR tyrosine kinase inhibitor, osimertinib, and exerted efficacy also in osimertinib-resistant PDX model. The preclinical assessment of safety in cynomolgus monkeys further revealed DB-1310 to have a good safety profile with a highest non severely toxic dose (HNSTD) of 45 mg/kg.ConclusionsThese finding demonstrated that DB-1310 exerted potent antitumor activities against HER3 + tumors in in vitro and in vivo models, and showed acceptable safety profiles in nonclinical species. Therefore, DB-1310 may be effective for the clinical treatment of HER3 + solid tumors.

Abstract A087: BI KRASmulti, a first-in-class, orally bioavailable and direct inhibitor of diverse oncogenic KRAS variants drives tumor regression in KRAS G12V-driven preclinical models

Abstract Alterations of KRAS are found in approximately one in seven of all human tumors, making KRAS one of the most prevalent oncogenic drivers. Gain-of-function missense mutations in KRAS, leading to its aberrant activation, are found in ~90% of pancreatic cancer, ~40% of colorectal cancer and ~30% of lung adenocarcinoma, with KRASG12V mutations accounting for ~28%, 9% and 6% of the cases, respectively. The poor outcome associated with these tumor types, particularly pancreatic cancer, as well as the lack of targeted inhibitors for KRASG12V calls for the urgent need to identify therapies able to effectively address the KRASG12V mutant allele. BI 3706674 is a novel, potent and orally available small molecule inhibitor of the KRAS oncogene. BI 3706674 binds non-covalently to multiple KRAS mutant alleles, including KRASG12V, in the GDP-bound state, and blocks downstream oncogenic signalling. Here, we show that BI 3706674 has a strong anti-proliferative activity in a panel of KRASG12V mutant cancer cell lines in vitro, along with significant pharmacodynamic (PD) biomarker modulation (including inhibition of ERK1/2 phosphorylation and down-regulation of DUSP6 mRNA).In vivo, a twice daily oral dose of 30 mg/kg was well-tolerated, while inducing tumour regression in several mutant KRASG12V cell line-derived and patient-derived xenograft (CDX and PDX) models across multiple different human tumor types. We have selected different non-small cell lung cancer xenograft models for extensive PK/PD/efficacy analysis in vivo. Results of the ongoing analysis will be shared. Feedback activation of upstream signalling pathways including receptor tyrosine kinase, such as the EGFR, has been suggested to be able to limit the efficacy of GDP-KRAS targeting compounds in preclinical studies. These findings have been supported by clinical combination trials involving KRASG12C inhibitors such as Adagrasib and Sotorasib and anti-EGFR modalities. We demonstrate for the first time that combination with anti-EGFR antibodies (e.g., Cetuximab) potently enhances the response observed with BI 3706674 in settings of KRASG12V mutant pancreatic, colorectal, and lung cancer. The deeper response observed upon combination of BI 3706674 with Cetuximab across multiple xenograft models provides a strong rationale for the clinical investigation of this combination therapy. Moreover, we utilize an ex vivo organoid platform to rapidly identify novel drug combinations with BI 3706674 that can potentially translate to clinical trials. The KRASmulti inhibitor BI 3706674 is currently undergoing IND enabling studies. Single agent dose escalation will include patients with cancers harbouring KRASG12V mutations. Citation Format: David H Peng, Antonio Tedeschi, Lorenz Herdeis, Fabio Savarese, Francesca Rocchetti, Popow Johannes, Heinrich J Huber, Nicola Melillo, Jake Dickinson, Hitesh B Mistry, Birgit Wilding, Matthias Treu, Julian Fuchs, Joachim Broker, Tobias Wunberg, Fiorella Schischlik, Jesse Lipp, Vandhana Ramamoorthy, Joseph R Daniele, Scott Kopetz, Michael Kim, Don L Gibbons, Christopher P Vellano, Joseph R Marszalek, Timothy P Heffernan, Darryl McConnell, Mark Pearson, Norbert Kraut, Dorothea Rudolph. BI KRASmulti, a first-in-class, orally bioavailable and direct inhibitor of diverse oncogenic KRAS variants drives tumor regression in KRAS G12V-driven preclinical models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A087.

Abstract LB_C04: Novel orally bioavailable macrocycles that target cyclin A and B elicit antitumor activity in breast cancer patient-derived xenograft models

Abstract Background: Cyclin/cyclin-dependent kinase (CDK) complexes regulate the activity of RB1 and E2F to drive cell cycle progression while ensuring fidelity of DNA replication. The interaction between cyclins and a critical subset of their substrates, including RB1 and E2F, is mediated by a conserved short linear RxL motif. RxL peptides have been shown to inhibit substrate phosphorylation by CDK2/cyclin A (AACR 2022 poster #5379). Previously, we have shown that macrocycles that selectively inhibit RxL-mediated binding to cyclins A and B (RxL inhibitors) lead to growth inhibitory activity in multiple cancer cell lines, and result in tumor regression in small cell lung cancer (SCLC) and ovarian cancer xenograft models (AACR 2023 poster #1560). Here, we test the antitumor activity of CID-016 in patient-derived xenograft (PDX) models from breast cancer. Methods: Bioinformatic analyses using RNA sequencing (RNAseq) and whole exome sequencing (WES) data from 39 breast cancer cell lines was employed to identify biomarker profiles that associate with sensitivity to CID-016. The antiproliferative activity of CID-016 was assessed by GI50 after 4-8 days of treatment. Hallmark pathway scores were calculated by Gene Set Variation Analysis (GSVA) method using MSigDb Hallmark collection from cell lines and existing PDX models. Based on the calculated hallmark pathway score, triple negative breast cancer (TNBC) PDX models were selected to test the anti-tumor activity of CID-016. In vivo, NMRI-Foxn1nu/nu mice were implanted with tumor fragments in the lower flank and treated with vehicle or CID-016 oral formulation of 50-100 mg/kg BID or 100 mg/kg QD, respectively for at least 28 days. Tumor volume and body weight were measured biweekly. Pharmacodynamic (PD) analyses will be conducted after treatment with CID-016. Results: Treatment of the breast cancer cell line panel with CID-016 demonstrated greater potency by GI50 in TNBC compared to other breast cancer cell lines (p=0.009). Gene expression levels of CCNB1, CCNA2, CCNE1 as well as target hallmark scores for “E2F targets”, “G2M checkpoint” and “Mitotic spindle” were significantly higher in TNBC vs other breast cancer cell lines. Oral treatment with CID-016 resulted in tumor regression in at least one triple-negative breast cancer PDX model and was well tolerated with no significant body weight changes. In vivo PD analyses will be assessed after 5 days of treatment with CID-016. Conclusions: The selective cyclin A/B RxL inhibitor CID-016 exhibits potent antitumor activity in TNBC preclinical models, consistent with the proposed mechanism of RB1/E2F pathway dysregulation. Given their compelling characteristics, orally bioavailable RxL inhibitors are advancing as the only first-in-class orally bioavailable dual inhibitors of cyclins A and B into clinical development. Citation Format: Mariana Paes Dias, Li-Fen Liu, Bernard Levin, Cristina Molina, Marta Guzmán, Olga Rodríguez, Evelyn W. Wang, Violeta Serra. Novel orally bioavailable macrocycles that target cyclin A and B elicit antitumor activity in breast cancer patient-derived xenograft models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C04.

Assessment of KRAS G12C Target Engagement by a Covalent Inhibitor in Tumor Biopsies Using an Ultra-Sensitive Immunoaffinity 2D-LC–MS/MS Approach

KRAS is one of the most frequently mutated oncogenes, with KRAS G12C recently becoming an actionable target for small molecule intervention. GDC-6036 is an investigational KRAS G12C inhibitor that acts by irreversibly binding to the switch II pocket of KRAS G12C when in the inactive GDP-bound state, thereby blocking GTP binding and activation. Assessing target engagement is an essential component of clinical drug development, helping to demonstrate mechanistic activity, guide dose selection, understand pharmacodynamics as it relates to clinical response, and explore resistance. Here, we report the development of an ultra-sensitive approach for assessing KRAS G12C engagement. Immunoaffinity enrichment with a commercially available anti-RAS antibody was combined with a targeted 2D-LC-MS/MS technique to quantify both free and GDC-6036-bound KRAS G12C proteins. A KRAS G12C-positive non-small cell lung cancer xenograft model was dosed with GDC-6036 to assess the feasibility of this assay for analyzing small core needle biopsies. As predicted, dose-dependent KRAS G12C engagement was observed. To date, a sensitivity of 0.08 fmol/μg of total protein has been achieved for both free and GDC-6036-bound KRAS G12C with as little as 4 μg of total protein extracted from human tumor samples. This sub-fmol/μg level of sensitivity provides a powerful potential approach to assess covalent inhibitor target engagement at the site of action using core needle tumor biopsies from clinical studies.

Inhibitory effects of icotinib combined with antiangiogenic drugs in human non-small cell lung cancer xenograft models are better than single target drugs.

BackgroundThis study aimed to evaluate the inhibitory effects and potential mechanisms of icotinib combined with antiangiogenic drugs on lung adenocarcinoma in vivo.MethodsA total of 72 mouse xenograft models established with human lung adenocarcinoma cells (HCC827) were randomly divided into six groups, including control, icotinib (Ic), bevacizumab (Bev), recombinant human endostatin (En), Ic + Bev and Ic + En groups. Mouse weights and tumor volumes were measured regularly. Half of the nude mice in each group were sacrificed after 16 days of drug treatment. The remaining animals were observed for another 16 days without drug supply. Immunohistochemical staining was performed to detect microvessel density in tumor heart, liver, brain specimens from the nude mice and Ki67 expression. Differential expression of vascular endothelial growth factor (VEGFA) in tumor tissue specimens was determined by ELISA and Western blot.ResultsThe results showed that the combined drugs inhibited tumor growth more substantially compared with single drugs, without increasing the toxic effects. The antiangiogenesis effect of the combination was better than that of single drug treatment. In addition, both types of targeted drugs and combination medication not only significantly reduced microvessel density in the tumor tissue itself, but also had a certain impact on decreasing microvessel density in the liver. The combination decreased VEGFA and Ki‐67 amounts significantly more than icotinib or endostatin as a monotherapy.ConclusionsIcotinib combined with bevacizumab or rh‐endostatin has a stronger inhibitory effect on tumor growth than single‐target drug in vivo, with no additional side effects.

Abstract 955: Preclinical characterization of a novel SSEA-4-targeting antibody drug conjugate, OBI-998

Abstract Stage-specific embryonic antigen-4 (SSEA-4) ceramide, a globo-series hexasaccharide glycosphingolipid, has been reported to be a tumor-associated antigen. Here, we present a novel antibody-drug conjugate (ADC) targeting SSEA-4 to evaluate its potential as a therapeutic agent for cancer treatment. OBI-998 is an ADC comprising the humanized anti-SSEA-4 antibody (OBI-898) that is conjugated to the highly potent microtubule-disrupting agent monomethyl auristatin E (MMAE) through maleimide and PEGylated cleavable linkers. We demonstrated the specificity of the naked antibody to SSEA-4 by screening against a panel of 21 related carbohydrate antigens using ELISA. OBI-998 displayed potent cytotoxic activity against cells (SKOV3) with a high level of SSEA-4 expression at sub-nanomolar potency but no effect on the viability of cells (SKBR3) with negligible SSEA-4 expression. The bystander killing effect of OBI-998 was shown by the transferring of conditioned medium from SKOV3 cells treated with 1 or 5 nM of OBI-998 to SKBR3 cells. Furthermore, significant bystander effects were observed by co-culturing high (NCI-N87) and low (PANC-1/GFP) SSEA-4-expressing cell lines at different ratios in the presence of 5 or 10 nM of OBI-998. OBI-998 was found to be rapidly internalized into cancer cells within 5 minutes upon binding to its target on the cell surface by confocal microscopy. OBI-998 showed significant anti-tumor efficacy in multiple cancer cell–derived xenograft models at doses of 1, 3, and 10 mg/kg in a dose-dependent manner. More importantly, OBI-998 at a dose of 10 mg/kg showed complete tumor regression in an EGFR-triple mutation non–small cell lung cancer xenograft model, which is resistant to the current last-line tyrosine kinase inhibitor, osimertinib. Pharmacokinetic analysis of OBI-998 revealed that total antibody and the conjugated antibody exhibited similar pharmacokinetic profiles, suggesting OBI-998 is highly stable in vivo. The biodistribution study in HCC1428 tumor-bearing mice indicated that MMAE accumulated in the tumor site at a higher level compared with other blood-rich organs. In addition, the MMAE levels in tumors reached peak level at 24 hours post-treatment, which was much higher than the maximum levels in other organs, and the level was sustained for 168 hours with tumor-to-muscle ratio of 329. OBI-998 is a novel ADC targeting SSEA-4 that possesses desired properties such as high target specificity, rapid internalization, potent cytotoxicity, and significant bystander effects. Furthermore, OBI-998 showed a high level of deposition and a persistent presence of MMAE in tumors and significant anti-tumor efficacy in a variety of animal models. Taken together, these results support the further development of OBI-998 as a therapeutic agent for SSEA-4-targeting cancer therapy. Citation Format: I-Ju Chen, Chun-Chung Wang, Chi-Sheng Shia, Chung-Chen Su, Chi-Huan Lu, Hui-Wen Chang, Ping-Tzu Chiu, Yueh-Chin Wu, Ming-Tain Lai, Wei-Chien Tang, Hsin-Yi Tung, Ren-Yu Hsu. Preclinical characterization of a novel SSEA-4-targeting antibody drug conjugate, OBI-998 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 955.

P71.02 Estrogen Promotes Resistance to Bevacizumab Treatment in Non-Small Cell Lung Cancer (NSCLC) Xenograft Models

P71.02 Estrogen Promotes Resistance to Bevacizumab Treatment in Non-Small Cell Lung Cancer (NSCLC) Xenograft Models

Abstract 4024: NAMPT inhibitor decreases NAMPT capture by an antibody directed against the 5-phosphoribosyl-1-pyrophosphate-binding loop: Rationale for an NAMPT occupancy assay

Abstract Nicotinamide phosphoribosyl transferase (NAMPT) catalyzes the rate-limiting step of nicotinamide adenine dinucleotide (NAD) biosynthesis. As NAMPT inhibitors (NAMPTi) have been shown to be substrates of NAMPT, binding of NAMPTi to NAMPT results in the NAMPTi-phosphoribose adduct (RP) that binds tightly and strongly inhibits the NAMPT enzyme. Structural analyses performed in this study unveiled the proximity of the NAMPTi-RP complex to the phosphoribosyl diphosphate (PRPP)-binding loop within the NAMPT catalytic site. The PRPP-binding loop of NAMPT is subject to conformational flexibility. The PRPP-binding loop domain is oriented to the outer side of the NAMPT dimer and can potentially serve as an antigen-binding site for antibodies. Interestingly, we show that the NAMPTi-RP adduct bound to NAMPT decreases NAMPT capture by an antibody that is directed against the c-terminal 5-phosphoribosyl-1-pyrophosphate-binding loop in NAMPT. This finding was then used to explore cellular NAMPT occupancy. The NAMPTi-RP complex displays a sustained, cellular NAMPT occupancy that correlates with the inhibition of NAMPT activity. Moreover, a good correlation between NAMPT occupancy, NAD decrease, and NAMPTi efficacy was observed in-vivo in a NCI-H82 small cell lung cancer (SCLC) xenograft model. NAMPT-occupancy assay can be used in clinical settings to better define the optimal dose levels and dose regimen for effective NAMPT inhibition. Citation Format: Maysoun Shomali, Sandrine Hilairet, Marianne Duhamel, Frederico Nardi, Thomas Bertrand, Geraldine Penarier, Laurent Debussche, Monsif Bouaboula. NAMPT inhibitor decreases NAMPT capture by an antibody directed against the 5-phosphoribosyl-1-pyrophosphate-binding loop: Rationale for an NAMPT occupancy assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4024.

Preclinical Anticancer Activity of an Electron-Deficient Organoruthenium(II) Complex.

Ruthenium compounds have been shown to be promising alternatives to platinum(II) drugs. However, their clinical success depends on achieving mechanisms of action that overcome Pt-resistance mechanisms. Electron-deficient organoruthenium complexes are an understudied class of compounds that exhibit unusual reactivity in solution and might offer novel anticancer mechanisms of action. Here, we evaluate the in vitro and in vivo anticancer properties of the electron-deficient organoruthenium complex [(p-cymene)Ru(maleonitriledithiolate)]. This compound is found to be highly cytotoxic: 5 to 60 times more potent than cisplatin towards ovarian (A2780 and A2780cisR), colon (HCT116 p53+/+ and HCT116 p53-/-), and non-small cell lung H460 cancer cell lines. It shows no cross-resistance and is equally cytotoxic to both A2780 and A2780cisR cell lines. Furthermore, unlike cisplatin, the remarkable in vitro antiproliferative activity of this compound appears to be p53-independent. In vivo evaluation in the hollow-fibre assay across a panel of cancer cell types and subcutaneous H460 non-small cell lung cancer xenograft model hints at the activity of the complex. Although the impressive in vitro data are not fully corroborated by the in vivo follow-up, this work is the first preclinical study of electron-deficient half-sandwich complexes and highlights their promise as anticancer drug candidates.

Abstract C061: BT8009, a Bicycle Toxin Conjugate targeting Nectin-4, shows target selectivity, and efficacy in preclinical large and small tumor models

Abstract Small molecule toxin conjugates offer a novel approach to tumor antigen specific targeting, allowing reduced systemic exposures and efficient tumor penetration of cytotoxic payloads, compared to large antibody toxin conjugates. BT8009 is a Bicycle Toxin Conjugate (BTC®) in which a Nectin-4 binding Bicycle® (bicyclic peptide) is conjugated through an inert sarcosine spacer chain, and a cleavable linker, to the antimitotic toxin MMAE. Increased Nectin-4 expression has been reported in multiple tumor types (including bladder, breast, esophageal, colorectal, lung, ovarian and pancreatic cancers) and so represents an appropriate tumor binding target. Bicycles are small (1.5KDa) fully synthetic, structurally constrained peptides with the specificities and affinities akin to antibodies, and pharmacokinetic properties more like small molecules. These features make Bicycles very attractive novel drug modalities for therapeutic approaches in which low systemic exposures coupled with high local drug concentrations are desired. We have previously described the identification and optimization process leading to selection of BT8009 as a candidate for IND enabling studies. BT8009 has low nanomolar (3nM) affinity for Nectin-4 and high selectivity (>1000 fold) over Nectins 1-3 and the 5 nectin-like family members (Necl1-5). BT8009 is active in a broad range of Nectin-4 positive cell line and patient derived xenograft tumors in vivo, leading to stable diseases and tumor regressions with durable responses. This abstract describes recent work characterizing this molecule. Selectivity of the Bicycle has been further demonstrated by assessing the binding of the biotinylated version of the Nectin-4 Bicycle on a human, plasma membrane protein cell array expressing 5528 full length human plasma membrane and secreted proteins. In this assay the Bicycle bound only Nectin-4 itself, again demonstrating the excellent target specificity. Efficacy data for BT8009 has now been extended to demonstrate the anti-tumor activity of BT8009 in large (1000mm3) xenograft tumors, in which rapid and near complete responses are observed. We have further characterized the efficacy profile of BT8009 in a panel of patient-derived non-small cell lung cancer (NSCLC) xenograft models, as well as in a panel of patient-derived pancreatic ductal (PDAC) xenograft models demonstrating a high frequency of stable diseases and partial responses. We will also describe the ongoing work for identification and validation of biomarkers for treatment response. BT8009 shows excellent efficacy in xenograft models expressing Nectin-4 target and its pharmacokinetic profile enables it to provide a rapid attainment of high tumor exposure levels with reduced systemic exposure. IND-enabling studies are currently ongoing with BT8009. Citation Format: Michael Rigby, Gavin Bennett, Liuhong Chen, Gemma Mudd, Paul Beswick, Helen Harrison, Sophie Watcham, Heather Allen, Amy Brown, Katerine Van Rietschoten, Philip Jeffrey, Peter U Park, Eric Haines, Nicholas Keen, Johanna Lahdenranta. BT8009, a Bicycle Toxin Conjugate targeting Nectin-4, shows target selectivity, and efficacy in preclinical large and small tumor models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C061. doi:10.1158/1535-7163.TARG-19-C061

DS-1205b, a novel selective inhibitor of AXL kinase, blocks resistance to EGFR-tyrosine kinase inhibitors in a non-small cell lung cancer xenograft model

The AXL receptor tyrosine kinase is involved in signal transduction in malignant cells. Recent studies have shown that the AXL upregulation underlies epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) resistance in EGFR-mutant non-small cell lung cancer (NSCLC). In this study, we investigated the effect of DS-1205b, a novel and selective inhibitor of AXL, on tumor growth and resistance to EGFR TKIs. In AXL-overexpressing NIH3T3 cells, DS-1205b potently inhibited hGAS6 ligand-induced migration in vitro and exerted significant antitumor activity in vivo. AXL was upregulated by long-term erlotinib or osimertinib treatment in HCC827 EGFR-mutant NSCLC cells, and DS-1205b treatment in combination with osimertinib or erlotinib effectively inhibited signaling downstream of EGFR in a cell-based assay. In an HCC827 EGFR-mutant NSCLC xenograft mouse model, combination treatment with DS-1205b and erlotinib significantly delayed the onset of tumor resistance compared to erlotinib monotherapy, and DS-1205b restored the antitumor activity of erlotinib in erlotinib-resistant tumors. DS-1205b also delayed the onset of resistance when used in combination with osimertinib in the model. These findings strongly suggest that DS-1205b can prolong the therapeutic benefit of EGFR TKIs in nonclinical as well as clinical settings.

Abstract CT019: Population pharmacokinetics of an aurora kinase A inhibitor, LY3295668 erbumine (AK-01), in patients with locally advanced or metastatic solid tumors

Abstract Background: The aurora kinase family plays a vital regulatory role in mitotic and meiotic events, with aurora kinase A (AurA) critical to centrosome maturation, mitotic spindle formation and checkpoint activation. LY3295668 erbumine (AK-01) is an inhibitor of AurA with 1000-fold selectivity for inhibition of AurA over aurora kinase B (AurB). Pharmacokinetic-pharmacodynamic (PK-PD) nonclinical models have shown that 90% inhibition of AurA phosphorylation (pAurA) over an extended period (≥ 16 hours per day) was required to yield efficacy in H446 (small cell lung cancer) xenograft models. Nonclinical plasma LY3295668 concentrations associated with 90% pAurA inhibition (IC90) were estimated previously using a direct sigmoidal relationship between PK and pAurA. In this first-in-man study, PK of LY3295668 in patients with advanced/metastatic cancer were evaluated. Human PK profiles were simulated subsequently and compared to pAurA IC90 to estimate a human efficacious dose range. Methods: LY3295668 erbumine was administered to 13 patients with locally advanced or metastatic solid tumors over a dose range of 25 to 75 mg given twice daily (BID). In the first cycle, serial plasma PK samples were collected up to 8 hours on Day 1 and Day 15. Additional trough samples were obtained prior to dosing on Day 2, on Day 8 and at discontinuation if applicable. PK parameter estimations were performed using non-linear mixed effects models in NONMEM v7.3. Results: The population PK model was developed using 146 plasma concentrations collected from 13 patients. A 2-compartment PK model with first order absorption best described the disposition of LY3295668. Following oral administration, LY3295668 was rapidly absorbed with peak concentrations occurring within 1 to 2 hours. The estimated elimination half-life was approximately 21 hours; therefore, steady state was attained within 4 to 5 days of dosing. Simulations of plasma concentration-time profiles showed that at the maximum tolerated dose (MTD) of 25 mg BID, 90% of patients are expected to achieve steady-state plasma concentrations greater than the pAurA IC90 for the entire day. Patients with dose limiting toxicities (DLTs) had the highest model-predicted exposures amongst the 13 patients. Conclusion: At the MTD of 25 mg BID, steady state LY3295668 plasma concentrations are maintained above the pAurA IC90 for the entire dosing interval, exceeding the minimum requirements for efficacy associated with non-clinical xenograft models. Trial Registration: NCT03092934 Citation Format: Eunice Yuen, Yu-Hua Hui, Amparo de la Peña, Sonya C. Tate, John R. Stille, Andrew Lithio, Patricia S. Smith, Quincy Chu, Gerald Batist, Nathaniel Bouganim, Caroline Fortier, Sara Zaknoen, Jill Kremer. Population pharmacokinetics of an aurora kinase A inhibitor, LY3295668 erbumine (AK-01), in patients with locally advanced or metastatic solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT019.

18F-Flortanidazole Hypoxia PET Holds Promise as a Prognostic and Predictive Imaging Biomarker in a Lung Cancer Xenograft Model Treated with Metformin and Radiotherapy.

Metformin may improve tumor oxygenation and thus radiotherapy response, but imaging biomarkers for selection of suitable patients are still under investigation. First, we assessed the effect of acute metformin administration on non-small cell lung cancer xenograft tumor hypoxia using PET imaging with the hypoxia tracer 18F-flortanidazole. Second, we verified the effect of a single dose of metformin before radiotherapy on long-term treatment outcome. Third, we examined the potential of baseline 18F-flortanidazole as a prognostic or predictive biomarker for treatment response. Methods: A549 tumor-bearing mice underwent a 18F-flortanidazole PET/CT scan to determine baseline tumor hypoxia. The next day, mice received a 100 mg/kg intravenous injection of metformin. 18F-flortanidazole was administered intravenously 30 min later, and a second PET/CT scan was performed to assess changes in tumor hypoxia. Two days later, the mice were divided into 3 therapy groups: controls (group 1), radiotherapy (group 2), and metformin + radiotherapy (group 3). Animals received saline (groups 1-2) or metformin (100 mg/kg; group 3) intravenously, followed by a single radiotherapy dose of 10 Gy (groups 2-3) or sham irradiation (group 1) 30 min later. Tumor growth was monitored triweekly by caliper measurement, and tumor volume relative to baseline was calculated. The tumor doubling time (TDT), that is, the time to reach twice the preirradiation tumor volume, was defined as the endpoint. Results: Thirty minutes after metformin treatment, 18F-flortanidazole demonstrated a significant change in tumor hypoxia, with a mean intratumoral reduction in 18F-flortanidazole tumor-to-background ratio (TBR) from 3.21 ± 0.13 to 2.87 ± 0.13 (P = 0.0001). Overall, relative tumor volume over time differed across treatment groups (P < 0.0001). Similarly, the median TDT was 19, 34, and 52 d in controls, the radiotherapy group, and the metformin + radiotherapy group, respectively (log-rank P < 0.0001). Both baseline 18F-flortanidazole TBR (hazard ratio, 2.0; P = 0.0004) and change from baseline TBR (hazard ratio, 0.39; P = 0.04) were prognostic biomarkers for TDT irrespective of treatment, and baseline TBR predicted metformin-specific treatment effects that were dependent on baseline tumor hypoxia. Conclusion: Using 18F-flortanidazole PET imaging in a non-small cell lung cancer xenograft model, we showed that metformin may act as a radiosensitizer by increasing tumor oxygenation and that baseline 18F-flortanidazole shows promise as an imaging biomarker.

Suppressing autophagy enhances disulfiram/copper-induced apoptosis in non-small cell lung cancer

Autophagy, a cellular survival mechanism, is thought to allow the recycling of cellular breakdown products when cancer cells are subjected to chemotherapy, thus decreasing drug-induced apoptosis. Disulfiram (DSF), a drug widely used to control alcoholism, possesses anticancer activity by inducing apoptosis in vitro and in vivo in a copper (Cu)-dependent manner. Our previous studies proved that DSF/Cu exerts increased anti-tumor effects on non-small cell lung cancer (NSCLC) xenograft models, and inhibits NSCLC recurrence driven by ALDH-positive cancer stem cells. The present study is designed to investigate whether DSF/Cu can induce autophagy in NSCLC cells and to determine the relationship between autophagy and apoptosis. First, we observed that DSF/Cu induced significant cytotoxicity and caspase-dependent apoptosis in NSCLC cells, accompanied by the formation of vacuoles in the cytoplasm. Next, we investigated the levels of autophagic markers, including LC3II (microtubule-associated protein 1 light chain 3), ATG5 and p62 with or without chloroquine by Western blot. In addition, we observed co-localization of LC3 and the lysosomal protein LAMP2 (lysosome-associated membrane protein 2) after treatment with DSF/Cu. The results showed that DSF/Cu induced autophagy. Finally, we demonstrated that DSF/Cu-induced apoptosis was greatly enhanced when autophagy was suppressed with Atg5 siRNA or 3-MA in NSCLC cells. This synergistic effect of DSF/Cu and 3-MA was further confirmed in the NSCLC xenograft model. Taken together, our results show that DSF/Cu stimulates autophagy in NSCLC cells, which is an impediment to DSF/Cu-induced apoptosis.

Promiximab-duocarmycin, a new CD56 antibody-drug conjugates, is highly efficacious in small cell lung cancer xenograft models.

Small cell lung cancer (SCLC) is of a highly invasive and metastatic lung cancer subtype and there had not been effective targeted therapies. CD56, a cell surface marker highly expressed on most SCLC, is a promising therapeutic target for treatment of this aggressive cancer. In this study, we generated a novel anti-CD56 antibody named promiximab, characterized by high affinity, internalization and tumor specificity. Then, the promiximab was conjugated with a potent DNA alkylating agent duocarmycin via reduced interchain disulfides to yield the promiximab-Duocarmycin (promiximab-DUBA) conjugates. Mass spectrometry analysis showed promiximab-DUBA had an average DAR (Drug-to-Antibody Ratio) of about 2.04. In vitro, promiximab-DUBA exerted strong inhibitory effects on SCLC cell lines NCI-H526, NCI-H524 and NCI-H69, with IC50 values of 0.07 nmol/L, 0.18 nmol/L and 0.29 nmol/L, respectively. In vivo antitumor activity, promiximab-DUBA at the dose of 5 mg/kg and 10 mg/kg every three days with a total of three times were sufficient to induce sustained regression of NCI-H526 tumors over control treatment with promiximab. Mostly, no recurrence was observed until 65 days post treatment with promiximab-DUBA. In the NCI-H69 subcutaneous xenograft model, significant inhibition of tumor growth was also observed following administration of promiximab-DUBA at the dose of 5 mg/kg or 10 mg/kg. Moreover, body weight and histopathology of major organs (liver, spleen, heart, lung and kidney) showed no significant changes after treatment of promiximab-DUBA. In conclusion, promiximab-DUBA is highly efficacious in small cell lung cancer xenograft models, and provides a new immunotherapy approach for SCLC.

Abstract DDT02-04: A novel PRMT5 inhibitor with potent in vitro and in vivo activity in preclinical lung cancer models

Abstract PRMT5 is a type II methyltransferase that specifically adds methyl groups to arginine as a long-lasting post-translational modification. The PRMT5/MEP50 complex regulates a plethora of cellular processes, such as epigenetics and splicing, which are notable events involved in tumorigenesis. Although not frequently mutated or amplified in tumors, elevated PRMT5 protein levels in lung and hematologic cancers are correlated with poorer survival. The PRMT5 inhibitor JNJ-64619178 has been selected as a clinical candidate based on its high selectivity and potency (subnanomolar range) under different in vitro and cellular conditions, paired with favorable pharmacokinetics and safety properties. JNJ-64619178 binds into the SAM binding pocket and reaches the substrate binding pocket to inhibit PRMT5/MEP50 function in a time-dependent manner. Broad cell line panel profiling of JNJ-64619178 revealed a wide range of sensitivity, which is indicative of a genomic dependency instead of a general cytotoxic on-target consequence of PRMT5 inhibition. Further investigations indicate a synthetic lethal correlation between PRMT5 inhibition and key cancer driver pathways. JNJ-64619178, dosed orally (10 mg/kg, every day), showed selective and efficient blockage of the methylation of SMD1/3 proteins, which are crucial components of the spliceosome and substrates of PRMT5/MEP50. JNJ-64619178 also demonstrated tumor regression in a biomarker-driven human small cell lung cancer xenograft model (NCI-H1048) and prolonged tumor growth inhibition after dosing cessation. In rodent and nonrodent toxicology studies, a tolerated dose of JNJ-64619178 has been identified, with the observed toxicity consistent with on-target activity. In summary, JNJ-64619178 has a favorable preclinical package that supports clinical testing in patients diagnosed with lung cancer and hematologic malignancies. Citation Format: Dirk Brehmer, Tongfei Wu, Geert Mannens, Lijs Beke, Petra Vinken, Dana Gaffney, Weimei Sun, Vineet Pande, Jan-Willem Thuring, Hillary Millar, Italo Poggesi, Ivan Somers, An Boeckx, Marc Parade, Erika van Heerde, Thomas Nys, Carol Yanovich, Barbara Herkert, Tinne Verhulst, Marc Du Jardin, Lieven Meerpoel, Christopher Moy, Gaston Diels, Marcel Viellevoye, Wim Schepens, Alain Poncelet, Joannes T. Linders, Edward C. Lawson, James P. Edwards, Dushen Chetty, Sylvie Laquerre, Matthew V. Lorenzi. A novel PRMT5 inhibitor with potent in vitro and in vivo activity in preclinical lung cancer models [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 DDT02-04. doi:10.1158/1538-7445.AM2017-DDT02-04

Abstract 37: Novel antibody-drug conjugates targeting ADAM9-expressing solid tumors demonstrate potent preclinical activity

Abstract ADAM9, also known as MDC9 or meltrin-γ, is a member of the ADAM (a disintegrin and metalloproteinase) family of proteases, which have been implicated in cytokine and growth factor shedding, and cell migration. Dysregulation of ADAM9 has been implicated in tumor progression and metastasis, as well as pathological neovascularization. ADAM9 overexpression has been shown to correlate with poor prognosis in prostate, renal, and pancreatic cancers. Using an immunization approach in which antibodies were raised to fetal progenitor and stem-like cancer cell lines followed by screening on tumor and normal tissues, we identified ADAM9 as a promising cell surface tumor target. FFPE-IHC expression analysis revealed that ADAM9 is overexpressed in multiple solid tumor indications relative to corresponding normal tissues. The overexpression of ADAM9 in tumors coupled with its restricted expression in normal tissues make ADAM9 an attractive target for antibody-drug conjugate (ADC) therapy. Here, we describe two ADCs both of which are based on a high affinity anti-ADAM9 antibody to selectively target ADAM9-expressing tumors. The first ADC utilizes the maytansine-derived microtubule disruptor, DM4, linked via a hindered disulfide hydrophilic linker (sulfo-SPDB). The second ADC exploits an ultra-potent DNA alkylating payload, DGN549, which is conjugated to two engineered cysteines via a peptide linker. Both conjugates bound with similar subnanomolar affinity to ADAM9-expressing cells. In vitro cytotoxicity studies showed that anti-ADAM9 ADCs can kill a broad panel of ADAM9-positve tumor cell lines, including lung, pancreatic, renal, prostate, and colon tumor cell lines. In particular, the anti-ADAM9-DGN549 conjugate was extremely potent with IC50 values ranging from 0.1 to 65 pM and was at least 2 logs more active than a non-targeting conjugate. Surprisingly, efficient in vitro cytotoxicity was observed at ADAM9 expression levels as low as a few thousand cell surface receptors per cell. Consistent with their in vitro activity, both anti-ADAM9 ADCs displayed compelling anti-tumor activity in xenograft models. In a CaLu3 non-small cell lung cancer xenograft model, anti-ADAM9-DM4 induced tumor growth delay at a single 1.25 mg Ab/kg dose. In the same model, a single intravenous dose of 0.25 mg Ab/kg of the anti-ADAM9-DGN549 produced durable complete remissions in 8/8 mice. A non-targeting DGN549 ADC was inactive even when dosed at 10 times that of the anti-ADAM9 ADC, demonstrating that targeted delivery of DGN549 through ADAM9 binding is required for activity. These data demonstrate that anti-ADAM9 ADCs exhibit antitumor activity against a broad panel of ADAM9-positive malignancies and cause durable remissions in preclinical models at doses expected to be clinically achievable. Anti-ADAM9 ADCs represent a promising therapeutic strategy to target a wide range of ADAM9-expressing tumors. Citation Format: Stuart W. Hicks, Nicholas C. Yoder, Deryk Loo, Asli Muvaffak, Yinghui Zhou, Megan E. Fuller, Molly A. McShea, Marian Themeles, Katherine H. Mucciarone, Juniper A. Scribner, Bhaswati Barat, Thomas Sun, James Tamura, Francine Z. Chen, Kerry A. Donahue, Tom Chittenden. Novel antibody-drug conjugates targeting ADAM9-expressing solid tumors demonstrate potent preclinical activity [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 37. doi:10.1158/1538-7445.AM2017-37

Abstract 4183: A novel selective DNA-PK inhibitor, M3814, as a potential combination partner of Etoposide and Cisplatin in the therapy of lung cancer

Abstract M3814 is a potent and selective inhibitor of DNA-PK, one of the key cellular regulators of DNA damage induced by ionizing radiation or certain cytostatics used in the treatment malignant disease. One of these drugs, Etoposide, induces double strand breaks (DSB) in cellular DNA. DSBs are most difficult to repair and, if left unrepaired, can lead to induction of cell cycle arrest and/or apoptosis and ultimately cell death. DNA-PK plays a critical role in the repair of DSB via the non-homologous end-joining pathway. M3814 was tested for activity in combination with Etoposide in a panel of 98 cancer cell lines derived from lung cancer. A broad potentiation effect of DNA-PK inhibitor was observed in most cancer cell lines. As a rule, cell lines sensitive to Etoposide demonstrated increased sensitivity to the combination. M3814 did not show significant effect on cancer cell growth/viability in combination with Cisplatin compared to Cisplatin alone at concentrations that effectively inhibit DNA-PK activity. At the same time, M3814 did not negatively affect the antitumor activity of Cisplatin. The therapeutic effect of M3814 in combination with the standard of care (SoC) regimen of Etoposide and Cisplatin was tested in the human small cell lung cancer xenograft model, NCI-H520. Triple combination of M3814, Etoposide and Cisplatin resulted in increased efficacy compared to SoC treatment arm. Since myeloid and lymphoid suppression is one of the dose limiting toxicities of the SoC regimen in patients, the effect of the triple combination on myeloid and lymphoid blood cells was investigated in immunocompetent mice. Whereas the SoC treatment showed reduction of the myeloid and lymphoid compartments, the addition of M3814 did not additionally reduce these cells neither in the treatment nor the recovery phase. Our results warrant further investigations to explore the potential of the combination in the clinical setting. Citation Format: Christian Sirrenberg, Astrid Zimmermann, Thomas Grombacher, Lyubomir T. Vassilev, Lars Damstrup, Frank T. Zenke, Andree Blaukat. A novel selective DNA-PK inhibitor, M3814, as a potential combination partner of Etoposide and Cisplatin in the therapy of lung 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 4183. doi:10.1158/1538-7445.AM2017-4183