Robert T. Streeper

Development and antitumor activity of a BCL-2 targeted single-stranded DNA oligonucleotide

PNT100 is a 24-base, chemically unmodified DNA oligonucleotide sequence that is complementary to a region upstream of the BCL-2 gene. Exposure of tumor cells to PNT100 results in suppression of proliferation and cell death by a process called DNA interference. PNT2258 is PNT100 that is encapsulated in protective amphoteric liposomes developed to efficiently encapsulate the PNT100 oligonucleotide, provide enhanced serum stability, optimized pharmacokinetic properties and antitumor activity of the nanoparticle both in vivo and in vitro. PNT2258 demonstrates broad antitumor activity against BCL-2-driven WSU-DLCL2 lymphoma, highly resistant A375 melanoma, PC-3 prostate, and Daudi-Burkitt’s lymphoma xenografts. The sequence specificity of PNT100 was demonstrated against three control sequences (scrambled, mismatched, and reverse complement) all encapsulated in a lipid formulation with identical particle characteristics, and control sequences did not demonstrate antiproliferative activity in vivo or in vitro. PNT2258 is currently undergoing clinical testing to evaluate safety and antitumor activity in patients with recurrent or refractory non-Hodgkin’s lymphoma and additional studies are planned.

Abstract 3529: Effect of PNT2258, an anti-BCL2 DNA-interference drug, on tumor growth and immunological markers in mice and humans.

Background: The dysregulation of apoptosis is a defining characteristic of malignant cells where excessive concentration of BCL2 protein contributes to the anti-apoptotic phenotype, driving development and subsequent resistance to therapy. Chromosomal translocations, including the t(14;18) rearrangement, up-regulate BCL2 transcription, preventing tumor cell death in B-cell lymphomas. A new class of therapeutic agents, called DNA interference (DNAi) drugs, exert their therapeutic effect by selectively blocking the transcription of oncogenes. PNT2258 is the lead DNAi drug currently undergoing clinical evaluation in patients with hematological malignancies. PNT2258 contains PNT100, a single stranded, native sequence oligonucleotide targeted against BCL2, which is delivered to cancer cells in a protective liposomal transport system. We present data on the anti-tumor and immunomodulatory effects of PNT228 in mice compared with that observed in patients with advanced solid tumors treated with escalating doses of PNT2258. Material and Methods: Twenty-two patients received PNT2258 doses ranging from 1 to 150 mg/mˆ2 as part of a Phase I dose-escalation study in patients with advanced, treatment refractory solid tumors. Patient plasma specimens were analyzed using a 61-marker multiplex immunoassay. Balb/c and WSU-DLCL2 xenograft mice received PNT2258 or an encapsulated scrambled control (oligonucleotide) sequence at a dose of 20 mg/kg by intravenous infusion. Murine plasma was analyzed with a 37-marker multiplex immunoassay. Results: In the murine xenograft model, PNT2258 demonstrated sequence-specific anti-tumor and anti-BCL2 activity not observed with the scrambled control. Xenograft mice demonstrated a strong innate immune response that was very similar in magnitude for both PNT2258 and scrambled control. Balb/c mice exhibited a broad and relatively weak immune response to PNT2258 and a stronger response to the scrambled control suggesting activation of both adaptive and innate immune responses. In patients, PNT2258 did not produce clinical signs of immune stimulation. Multiplex immunoassay revealed a lack of significant drug-induced modulation of inflammatory biomarkers following treatment. PNT2258 did induce statistically significant dose-dependent changes in IP-10, leptin, MIP-1β, MCP-1, IL-17F, and IL-1RA consistent with a mechanistic response to BCL2 suppression. Conclusions: Biomarker response profiles of encapsulated human sequence-specific oligonucleotide therapeutics in mice are not predictive of responses in humans. PNT2258 is safe and well tolerated in patients with no evidence of an innate (TLR) response. The observed biomarker profile provides mechanistic confirmation of drug-induced BCL2 suppression at all doses tested. Leptin represents a unique biomarker that may be used to monitor PNT2258 anti-BCL2 activity in the clinic. Citation Format: Elzbieta Izbicka, Robert Streeper, Michael J. Wick, Drew Rasco, Amita Patnaik, Kyriakos P. Papadopoulos, Anthony W. Tolcher, Shari Gaylor, Michael J. Woolliscroft, Richard A. Messmann, Wendi V. Rodrigueza. Effect of PNT2258, an anti-BCL2 DNA-interference drug, on tumor growth and immunological markers in mice and humans. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3529. doi:10.1158/1538-7445.AM2013-3529

Abstract 1733: Development of a novel blood plasma protein test for diagnosis of lung cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: A blood test for detection of lung cancer (LC) could provide survival benefits in high-risk populations. We conducted a retrospective study as the next step of our effort to develop a test for diagnosis of non-small cell lung carcinoma (NSCLC), the most common form of LC, by measuring levels of protein biomarkers in plasma. Materials and Methods: We quantified levels of 106 proteinaceous biomarkers using multiplexed immunoassays of subjects a) diagnosed with stage I/II NSCLC, b) normal non-smoking controls, and c) normal heavy smoking controls (> 10 p.y.). All groups consisted of Caucasian men and women (93 subjects each, ages range 45-87, Ave 59). The specimens were randomized and tested in a blinded manner. A support vector machine (SVM) algorithm, developed and tested in a pilot study, was used to identify differentially expressed biomarkers in the disease and control groups taking into account patients’ gender. Pathway analysis was applied to characterize pathology- and gender-specific patterns of biomarker expression. Results: In a recent pilot study we developed SVM models that classified subjects to NSCLC or normal using 59 plasma markers or subsets thereof, for both genders combined or separately. When data for both genders were analyzed together, SVM classified subjects to NSCLC or normal and identified 4 differentially expressed markers: EGF, sCD40 ligand, IL-8 and MMP-8; sensitivity (SE) 0.93 (95% CI: 0.88-0.96), specificity (SP) 0.87 (95% CI: 0.80-0.92). Considering the sexes separately, the differentially expressed best subset of markers for men (EGF, IL-8, sFAS, MMP-9 and PAI-1) had both SE and SP of 1.0. For women, the best subset of markers (EGF, sCD40 ligand and IL-8) also yielded SE and SP of 1.0. The present study has further confirmed and refined our earlier findings and expanded panels of diagnostic biomarkers for NSCLC with comparable sensitivities and specificities. Analysis of cellular pathways represented by the discriminatory biomarker signatures suggested a role of sex hormone-driven differences of NSCLC plasma biomarkers. Conclusions: The set of diagnostic biomarkers for NSCLC identified in this study holds significant promise for use in the early diagnosis of lung cancer and warrants validation of findings in other ethnic groups and for potentially confounding diseases in clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1733. doi:1538-7445.AM2012-1733

Abstract B2: Patterns of serum biomarkers for lung cancer are different for men and women

Introduction: Lung cancer has one of the highest mortality rates among all human cancers. Higher mortality has been noted in the past in men with the history of smoking but no significant differences in mortality exist in never smoking men and women. Some gender differences to date have been linked to genes located on sex chromosomes. Methods: We quantified levels of 57 cytokines, chemokines and growth factors using multiplexed immunoassays to determine patterns of serum biomarkers in men and women with non-small cell lung cancer (NSCLC; 250 men, 116 women) and compared these with serum marker patterns in subjects with asthma (AST; 68 men, 132 women) and normal controls (NOR; 250 men, 116 women). Data were reduced using inter-pathology comparisons with statistical significance determined using the Kruskall-Wallis U test with p Results: The markers showed unique gender- and disease specific patterns. For unisex analysis of NSCLC, we identified 36 markers with the 25% change cutoff threshold and 32 markers with the 50% cutoff. For women, we found 32 markers with the 25% cutoff and 30 with the 50% cutoff. For men, 39 markers were found at the 25% cutoff and 37 at the 50% cutoff. Expression of four markers was unique for women with NSCLC compared to NOR: IL-8 and serum amyloid P (downregulated), serum amyloid A and C-reactive protein (all upregulated). Five markers were unique for men with NSCLC compared to NOR: Insulin (downregulated), matrix metalloproteinases-7 and -8, resistin and hepatocyte growth factor (all upregulated). Three markers showed opposite patterns of expression; VEGF was downregulated in women and upregulated in men with NSCLC compared to NOR. Leptin was upregulated in women and downregulated in men and MIP-1α were upregulated in men and downregulated in women with NSCLC compared to NOR. Conclusion: Our study has revealed surprisingly large qualitative and quantitative differences in biomarker expression patterns and levels in serum specimens from men and women suffering from NSCLC. The markers appear to be products of genes residing on several chromosomes and are not limited to sex chromosomes. Our findings may have implications for the diagnosis of non-small cell lung cancer, early detection of the disease through simple blood tests, characterization of disease progression and differentiation among the pathologies. It may find application in discovery and development of novel therapeutic strategies for human diseases. Citation Information : Clin Cancer Res 2010;16(7 Suppl):B2

Abstract C15: A novel panel of serum biomarkers distinguishes asthma from non‐small cell lung cancer

Purpose: Asthma affects an increasing proportion of the population and may predispose sufferers to lung cancer. Frequent misdiagnosis of asthma and lack of early detection tests for lung cancer might be mitigated by better diagnostic tests. We used multiplexed immunoassays and mass spectrometry to identify serum biomarkers capable of distinguishing asthma from non‐small cell lung cancer, the most common type of lung cancer. Methods: Sera from normal subjects (NO; n = 300), patients with asthma (AST; n=180), and non‐small cell lung cancer (LC; n = 334) were acquired from commercial vendors. Levels of 58 cytokines, chemokines and growth factors were quantified using multiplexed immunoassays. For biomarker discovery, specimens were digested with trypsin and analyzed by liquid chromatography electrospray ionization MS (LC ESI MS/MS). Proteins were identified using Mascot search software. Validation of select biomarkers identified by MS was achieved by immunodetection of target proteins in serum specimens. Data were reduced using inter‐pathology comparisons with statistical significance determined using Student9s t‐test. Results: Multiplexed immunoassays identified 29 analytes with significant (t Conclusion: We have identified a group of serum biomarkers having high inter‐pathology discrimination power that are capable of differentiating AST and LC from normal controls and that may potentially be used in diagnostic tests for early detection of lung pathologies. Our experimental strategy is widely applicable to discovery and validation of biomarkers for diverse human diseases, response to a therapy, or pre‐selection of patients for clinical trials. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C15.