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Background Adaptor proteins such as growth factor receptor-bound protein-2 (Grb2) play important roles in cancer cell signaling. In the present study, we examined the biological effects of liposomal antisense oligodeoxynucleotide that blocks Grb2 expression (L-Grb2) in gynecologic cancer models. Materials and methods Murine orthotopic models of ovarian (OVCAR5 and SKOV3ip1) and uterine (Hec1a) cancer were used to study the biological effects of L-Grb2 on tumor growth. In vitro experiments (cell viability assay, Western blot analysis, siRNA transfection, and reverse phase protein array) were carried out to elucidate the mechanisms and potential predictors of tumor response to L-Grb2. Findings Treatment with L-Grb2 decreased tumor growth and metastasis in orthotopic models of ovarian cancer (OVCAR5, SKOV3ip1) by reducing angiogenesis and increasing apoptosis at a dose of 15 mg/kg with no effect on mouse body weight. Treatment with L-Grb2 and paclitaxel led to the greatest decrease in tumor weight (mean ± SEM, 0.17 g ± 0.10 g) compared with that in control mice (0.99 g ± 0.35 g). We also observed a reduction in tumor burden after treatment with L-Grb2 and the anti-VEGF antibody B-20 (86% decrease in tumor weight compared with that in controls). Ovarian cancer cells with ErbB2 amplification (OVCAR8 and SKOV3ip1) were the most sensitive to Grb2 downregulation. Reverse phase protein array analysis identified significant dysregulation of metabolites (LDHA, GAPDH, and TCA intermediates) in ovarian cancer cells after Grb2 downregulation. Interpretation L-Grb2 has therapeutic efficacy in preclinical models of ovarian and uterine cancer. These findings support further clinical development of L-Grb2.

Background: STAT3 is a transcription factor that regulates various tumorigenic processes, such as tumor proliferation, metastasis, and drug resistance. Its overexpression and aberrant activation characterize many cancers (including breast, lung, ovarian, liver and colon cancer). Metastasis and drug resistance are two main causes of patient mortality. Activation of the JAK-STAT3 and the FGFR1-STAT3 pathways in breast and ovarian cancer cells promotes tumor initiation, migration, and taxol resistance. STAT3 also contributes to epithelial-mesenchymal transition and promotes 5-FU resistance in colorectal cancer cells. Its role in numerous malignancies has made STAT3 an ideal cancer therapeutic target. BP1003 is a neutral DOPC liposome incorporated with a nuclease-resistant P-ethoxy antisense oligodeoxynucleotide targeting STAT3 mRNA. Here we determine the effects of BP1003 in combination with taxol and 5-FU on breast and ovarian cancer cells. Methods: Western blots determined the extent by which BP1003 reduced STAT3 protein levels. The effect of sequential taxol or 5-FU (5 h pre-treatment) and BP1003 treatments (96 h) on cell viability was analyzed with resazurin-based assays. Migration and colony formation assays were performed to assess the metastatic potential of treated cells. Cells pre-treated with BP1003 (96 h) and taxol (2 h) were placed in chemotaxis chambers and allowed to migrate for 5-18 h before quantification. Colony formation involved the treatment of a low number of cells with chemotherapeutic drugs for 24 h prior to the addition of BP1003 for 10-14 days. Tumor spheroids were used to mimic in vivo solid tumor growth. Three days after formation, spheroids were treated with chemotherapeutic drugs for 1 day before being treated with BP1003 for 3 additional days. Results: Treatment of BT549, SK-Br-3 and SK-Ov-3 cells with 250 μg/ml of BP1003 resulted in a 30%-50% decrease in STAT3 expression. Cell viability decreased by 20-35% with 200 μg/ml of BP1003 and by 45-60% in combination with taxol or 5-FU. BP1003 in combination with taxol also decreased the colony formation potential of BT549 cells by 20%. Migration of BT549 cells was reduced by 30% with 200 μg/ml BP1003 alone and by 50% in combination with taxol. The area of BT549 and SK-Ov-3 spheroids under BP1003 and taxol combination treatments was 40%-50% smaller than control spheroids and 20%-30% smaller than taxol mono-treatment. Conclusions: BP1003 efficiently reduces STAT3 expression and enhances the sensitivity of breast and ovarian cancer cells to taxol and 5-FU. These results are in line with previous work in which BP1003 + gemcitabine displayed enhanced anti-tumor activity in pancreatic ductal adenocarcinoma. Together these results strongly suggest that BP1003 combination therapy is a novel strategy for patients with advanced solid tumors. Citation Format: Maria Gagliardi, Ana Tari Ashizawa. Targeting STAT3 with novel liposome-incorporated antisense oligonucleotide technology enhances the efficacy of paclitaxel (taxol) and 5-fluorouracil (5-FU) in breast and ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3285.

Cerebellar Purkinje cells (PCs) show conspicuous damages in many ataxic disorders. Targeted delivery of short nucleic acids, such as antisense oligonucleotides, to PCs may be a potential treatment for ataxic disorders, especially spinocerebellar ataxias (SCAs), which are mostly caused by a gain of toxic function of the mutant RNA or protein. However, oligonucleotides do not cross the blood-brain barrier (BBB), necessitating direct delivery into the central nervous system (CNS) through intra-thecal, intra-cisternal, intra-cerebral ventricular, or stereotactic parenchymal administration. We have developed a novel liposome (100 to 200 nm in diameter) formulation, DCL64, composed of dipalmitoyl-phosphatidylcholine, cholesterol, and poloxamer L64, which incorporates oligonucleotides efficiently (≥ 70%). Confocal microscopy showed that DCL64 was selectively taken up by brain microvascular endothelial cells by interacting with low-density lipoprotein receptor (LDLr) family members on cell surface, but not with other types of lipid receptors such as caveolin or scavenger receptor class B type 1. LDLr family members are implicated in brain microvascular endothelial cell endocytosis/transcytosis, and are abundantly localized on cerebellar PCs. Intravenous administration of DCL64 in normal mice showed distribution of oligonucleotides to the brain, preferentially in PCs. Mice that received DCL64 showed no adverse effect on hematological, hepatic, and renal functions in blood tests, and no histopathological abnormalities in major organs. These studies suggest that DCL64 delivers oligonucleotides to PCs across the BBB via intravenous injection with no detectable adverse effects. This property potentially makes DCL64 particularly attractive as a delivery vehicle in treatments of SCAs.

Background: About two-thirds of AML patients harbor mutations in signaling and kinase pathways (e.g., FLT3, KRAS, NRAS, PTPN11, NF1, KIT), which utilize the growth factor receptor bound protein-2 (Grb2) to induce AML progression. Prexigebersen (BP1001), a liposome-incorporated Grb2 antisense oligonucleotide that inhibits Grb2 expression, suppresses cancer growth and survival. A Phase I/IB single center study (Ohanian, Lancet Haematol 2018) in relapsed/refractory AML patients demonstrated BP1001 safety up to 90 mg/m 2 and demonstrated CR/CRi in 3 of 6 relapsed/refractory AML patients treated with BP1001 + low dose cytarabine combination in the Phase IB portion. Preclinical studies indicated that BP1001 enhanced the inhibitory effects of decitabine (DAC), or DAC + venetoclax (VEN) against AML cells. Thus, we designed a clinical trial with three treatment arms: (1) BP1001 + DAC + VEN for newly diagnosed AML, (2) BP1001 + DAC + VEN for relapsed/refractory AML, (3) BP1001 + DAC for relapsed/refractory AML patients who are VEN-resistant or -intolerant [ClinicalTrials.gov Identifier: NCT02781883]. The study was amended to obtain safety run-in data for patients treated with BP1001 + DAC first before proceeding to safety run-in for patients treated with BP1001 + DAC + VEN. Here we report the safety run-in and efficacy data of AML patients treated with BP1001 + DAC or BP1001 + DAC + VEN. Methods: This is a multi-center open-label, Phase II study in newly diagnosed and relapsed/refractory AML patients to determine the safety and preliminary efficacy of BP1001 (given at 60 mg/m 2 over 2-3 h twice weekly for a total of 8 doses over a 28-day cycle) in combination with DAC (20 mg/m 2, once daily for 5 days) or in combination with DAC and VEN (400 mg, once daily). Eligible patients were considered unsuitable for or refused intensive chemotherapy and had ECOG performance status of 0-2. Patients treated with at least 1 cycle of therapy were evaluable for safety run-in. Results: Between April 2018 and May 2020, 6 patients (4 male: 67%) with median age 72 years (range, 51 - 76) were treated with at least 1 cycle of BP1001 + DAC combination therapy. Four patients (67%) had de novo AML and two (33%) had secondary AML. All patients in this cohort were considered high risk due to having either adverse risk status by ELN (n=5) or treated secondary AML (n=1) (transformed from myelodysplastic syndrome/myeloproliferative neoplasm after failing 4 lines of prior therapy). Adverse events (AEs) were generally consistent with those expected with DAC and/or AML and included diarrhea (67%), fatigue (50%), constipation (50%), nausea (50%), dyspnea (50%), hypoxia (50%), hypotension (50%) and pain in extremity (50%). The most frequent serious AE (SAE) was bacteremia (33%). Two de novo patients (33%) achieved a CRi and 1 treated secondary AML patient (17%) achieved a PR. Patients received a median of 2.5 cycles of therapy (range, 1 - 6); median duration of follow-up was 140 days (range, 72 - 678). Since May 2020, 6 patients (2 male: 33%) with median age 61 years (range, 44 - 81) were treated with at least 1 cycle of BP1001 + DAC + VEN combination therapy. Two (33%) had de novo AML, and four (67%) were relapsed/refractory. All patients in this cohort were adverse-risk by ELN (n=2) or relapsed/refractory (n=4). AEs were generally as expected with DAC, VEN and/or AML, including fatigue (67%), diarrhea (67%), constipation (67%), decreased appetite (67%), hypokalemia (67%), hypocalcemia (50%), edema peripheral (50%), asthenia (50%), nausea (50%), abdominal pain (50%), dysphagia (50%), hypotension (50%) and febrile neutropenia (50%). The most frequent SAEs were sepsis (33%) and febrile neutropenia (50%). Four patients (67%) achieved a CR/CRi/MLFS (n=1/2/1) and 1 (17%) achieved a PR. Of these patients, 3 were relapsed/refractory (1 CR/1 CRi/1 MLFS) and two were de novo (1 CRi/1 PR). Patients received a median of 4 cycles of therapy (range, 1 - 9); median duration of follow-up was 177 days (range, 113 - 318). Conclusions: BP1001-based combination therapy has been safely administered to high-risk and relapsed/refractory AML patients who were considered unsuitable for standard chemotherapy. Preliminary data showed the combination treatment of BP1001 + DAC or BP1001 + DAC + VEN was well-tolerated, with encouraging efficacy signals. The study will continue enrollment across all three cohorts. Drug dosing for the study will proceed as planned. Figure 1 Figure 1. Disclosures Lin: AbbVie, Aptevo Therapeutics, Astellas Pharma, Bio-Path Holdings, Celgene, Celyad, Genentech-Roche, Gilead Sciences, Incyte, Jazz Pharmaceuticals, Novartis, Ono Pharmaceutical, Pfizer, Prescient Therapeutics, Seattle Genetics, Tolero, Trovagene: Research Funding. Ritchie: Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Incyte: Consultancy, Honoraria, Speakers Bureau; ARIAD Pharmaceuticals: Ended employment in the past 24 months, Speakers Bureau; Novartis: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Celgene/BMS: Consultancy, Other: travel support, Speakers Bureau; Astellas: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; NS Pharma: Research Funding; Protaganist: Consultancy, Honoraria; Jazz: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding. Pemmaraju: Clearview Healthcare Partners: Consultancy; ASH Communications Committee: Membership on an entity's Board of Directors or advisory committees; Plexxicon: Other, Research Funding; MustangBio: Consultancy, Other; Roche Diagnostics: Consultancy; Springer Science + Business Media: Other; DAVA Oncology: Consultancy; Stemline Therapeutics, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Abbvie Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Celgene Corporation: Consultancy; LFB Biotechnologies: Consultancy; Protagonist Therapeutics, Inc.: Consultancy; HemOnc Times/Oncology Times: Membership on an entity's Board of Directors or advisory committees; Samus: Other, Research Funding; Daiichi Sankyo, Inc.: Other, Research Funding; Novartis Pharmaceuticals: Consultancy, Other: Research Support, Research Funding; Affymetrix: Consultancy, Research Funding; Blueprint Medicines: Consultancy; Cellectis S.A. ADR: Other, Research Funding; Aptitude Health: Consultancy; CareDx, Inc.: Consultancy; Dan's House of Hope: Membership on an entity's Board of Directors or advisory committees; Sager Strong Foundation: Other; ASCO Leukemia Advisory Panel: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy; Bristol-Myers Squibb Co.: Consultancy; ImmunoGen, Inc: Consultancy; Pacylex Pharmaceuticals: Consultancy. Borthakur: Ryvu: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; University of Texas MD Anderson Cancer Center: Current Employment; Astex: Research Funding; Protagonist: Consultancy; ArgenX: Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy; Takeda: Membership on an entity's Board of Directors or advisory committees. Tari Ashizawa: Bio-Path Holdings, Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Kantarjian: Immunogen: Research Funding; Ipsen Pharmaceuticals: Honoraria; AbbVie: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Daiichi-Sankyo: Research Funding; Aptitude Health: Honoraria; Precision Biosciences: Honoraria; Novartis: Honoraria, Research Funding; Taiho Pharmaceutical Canada: Honoraria; NOVA Research: Honoraria; Astra Zeneca: Honoraria; Ascentage: Research Funding; BMS: Research Funding; Astellas Health: Honoraria; Pfizer: Honoraria, Research Funding; Jazz: Research Funding; KAHR Medical Ltd: Honoraria. Cortes: Novartis: Consultancy, Research Funding; Sun Pharma: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Bristol Myers Squibb, Daiichi Sankyo, Jazz Pharmaceuticals, Astellas, Novartis, Pfizer, Takeda, BioPath Holdings, Incyte: Consultancy, Research Funding; Bio-Path Holdings, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Research Funding. Roboz: Amgen: Consultancy; MEI Pharma - IDMC Chair: Consultancy; Bayer: Consultancy; AstraZeneca: Consultancy; AbbVie: Consultancy; Celgene: Consultancy; Blueprint Medicines: Consultancy; Glaxo SmithKline: Consultancy; Agios: Consultancy; Helsinn: Consultancy; Jazz: Consultancy; Janssen: Consultancy; Astex: Consultancy; Bristol Myers Squibb: Consultancy; Actinium: Consultancy; Jasper Therapeutics: Consultancy; Novartis: Consultancy; Otsuka: Consultancy; Mesoblast: Consultancy; Daiichi Sankyo: Consultancy; Astellas: Consultancy; Janssen: Research Funding; Pfizer: Consultancy; Roche/Genentech: Consultancy.

Background: B-cell lymphoma 2 (Bcl-2) is an oncoprotein that promotes tumor cell survival and drug resistance. Elevated Bcl-2 expression in numerous human cancers has made it an ideal drug target. Venetoclax, the first FDA approved Bcl-2 inhibitor, is indicated for hematologic malignancies. Accompanying its promising clinical results, however, is the emergence of venetoclax resistance. A recent study found that AML patients who had relapsed from frontline venetoclax-based treatment were refractory to salvage therapy and had a median survival of less than 3 months. Thus, novel treatment approaches for such patients are urgently needed. BP1002, a non-toxic neutral dioleoylphosphatidylcholine liposome incorporating a nuclease-resistant P-ethoxy antisense oligonucleotide (oligo), was developed to target Bcl-2 mRNA. The safety and efficacy of BP1002 is being investigated in patients with advanced lymphoid malignancies. Venetoclax in combination with decitabine is an approved treatment for elderly AML patients. Here we determine the effects of BP1002 plus decitabine in venetoclax-resistant cells. Methods: Cell lines: Venetoclax-resistant hematologic cell lines, MV-4-11- R, SU-DHL-2-R, SU-DHL-6-R, were established from the parental cells by exposing the cells to increasing doses of venetoclax for 10-12 weeks.BP1002: A Bcl-2 antisense oligo, which targets the translation initiation site of human Bcl-2 mRNA, was incorporated in BP1002. Combination study: Parental and venetoclax-resistant cells were treated with decitabine for 2 days followed by the addition of BP1002 for another 4 days of incubation. Resazurin-based cell viability assays and Western blots were conducted to determine the inhibitory effects of BP1002 in parental and resistant cells. Results: The IC50 values of venetoclax in resistant cells were 3 to 40 times higher than those in parental cells. MV-4-11-R, SU-DHL-6-R, SU-DHL-2-R cells are resistant up to 0.1 μM, 0.8 μM, and 7 μM venetoclax, respectively. Compared to parental cells, venetoclax-resistant cells displayed 2- to 3-fold elevated levels of Bcl-2 protein and other anti-apoptotic proteins such as Mcl-1. BP1002 in combination with decitabine decreased the viability of parental cells. BP1002 reduced Bcl-2 levels in venetoclax-resistant cells. Also, the BP1002 plus decitabine combination was more potent than the venetoclax plus decitabine combination in reducing the viability of the resistant cells, by about 50-70%. Conclusions: Our data indicate that, despite expressing higher levels of anti-apoptotic proteins, venetoclax-resistant cells are sensitive to the BP1002 plus decitabine combination. These promising preclinical results suggest that the BP1002 plus decitabine combination is a potential treatment for patients who have relapsed from frontline venetoclax-based therapies. Citation Format: Maria Gagliardi, Ana Tari Ashizawa. The combination of liposomal Bcl-2 antisense oligonucleotide (BP1002) with decitabine is efficacious in venetoclax-resistant cells [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 939.

Background: Bcl-2 protein is essential for cancer cell survival. Inhibition of Bcl-2 expression may lead to cancer cell death. A nuclease-resistant, hydrophobic Bcl-2 antisense oligonucleotide (oligo) incorporated in a neutral dioleoylphosphatidylcholine liposome (L-Bcl-2, also known as BP1002) is utilized to inhibit Bcl-2 expression. Preclinical studies showed that L-Bcl-2 inhibited Bcl-2 protein expression and in vivo tumor growth. Bcl-2 expression is frequently altered in human cancers, including lymphomas. We postulate that BP1002 will have anti-tumor activity against lymphomas. This first-in-human BP1002 study is proposed as a dose-escalation monotherapy study in patients with advanced lymphoid malignancies. Methods: Subjects: BP1002 will be given to adult subjects diagnosed with refractory/relapsed chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, non-Hodgkin lymphoma (B-cell and T-cell) and Waldenströms macroglobulinemia. Study Design: The study will utilize a single-arm, sequential, dose-escalation design (3+3) to identify dose-limiting toxicities (DLTs), recommended Phase II dose (RP2D), pharmacokinetics (PK), pharmacodynamics, and efficacy of BP1002. BP1002 will be administered to subjects intravenously twice weekly per 28-day cycle (8 doses). There are 2 planned dose levels, 20 and 40 mg/m2. Successive cohorts of eligible patients will be treated with BP1002. A higher dose level cannot be opened until a minimum of three patients have completed an entire therapy cycle at the preceding dose level without having DLTs. Any DLT observed will trigger an expansion of a cohort from 3 to 6 patients. A second DLT at any dose level will identify the maximum dose. The dose below that will be the maximum tolerated dose (MTD). The RP2D will be determined using the MTD data and all information regarding safety, biological activity, PK and pharmacodynamics. Primary objectives: To determine • Safety and tolerability of escalating doses of BP1002 • RP2D of BP1002 • PK of BP1002 • BP1002 activity on Bcl-2 expression Secondary objectives: To obtain evidence of tumor response (overall response rate = complete remission + partial remission + minor response), and estimates for other response parameters (time to progression, progression-free survival, event-free survival) Exploratory objectives: To correlate treatment response with biological activity, cytogenetic and molecular characteristics Safety assessments will include identification of DLTs, serious adverse events, treatment-emergent adverse events and treatment-emergent laboratory abnormalities Response assessments will be determined per the 2018 International Workshop Group on CLL (iwCLL) response criteria, the International Working Group consensus response evaluation criteria in lymphoma (RECIL 2017), or the Eighth International Workshop for Waldenströms Macroglobulinemia response criteria. Citation Format: Ana Tari Ashizawa, Locke Bryan, William Wierda. A phase I clinical trial to study the safety, pharmacokinetics, and efficacy of BP1002 (L-Bcl-2) antisense oligonucleotide in patients with advanced lymphoid malignancies [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 CT240.

TPS7561 Background: The growth factor receptor bound protein-2 (Grb2) is vital to oncogene signaling and tumor progression. BP1001 is a liposome-incorporated Grb2 antisense oligonucleotide that inhibits Grb2 expression. Grb2 inhibition suppresses cancer growth and survival. A Phase I/Ib single center study in patients with refractory/relapsed leukemias demonstrated the safety of BP1001 up to 90 mg/m2 dose and the efficacy of BP1001 in combination with low dose cytarabine (LDAC) in refractory/relapsed acute myeloid leukemia (AML) patients (Ohanian, Lancet Haematol 2018). Based on pharmacokinetic (PK) considerations, the recommended Phase II dose was 60 mg/m2. A phase II study was initiated to explore the clinical impact of BP1001 in combination with LDAC in untreated AML patients. Interim safety and efficacy of BP1001 + LDAC in untreated AML patients was presented at the 2018 Annual ASH Meeting. BP1001 was safely administered to 25 patients with untreated AML, who were considered unfit for standard chemotherapy. Efficacy data compared favorably to what has been reported with available options for unfit patients largely with secondary AML or adverse-risk AML. Since the Interim Data presentation, the study was amended to investigate BP1001 + decitabine combination and include myelodysplastic syndrome (MDS) patients. This is because BP1001 enhanced decitabine anti-leukemic effects in preclinical studies. Methods: This is a multi-center, open-label study with 2 parallel cohorts of BP1001 + decitabine treatment in untreated AML/high risk MDS patients and refractory/relapsed AML/high risk MDS patients who are considered by the investigator unsuitable for or refused intensive chemotherapy. BP1001 is given intravenously (IV) at 60 mg/m2 twice weekly. Decitabine is given 20 mg/m2 IV daily for 5 consecutive days. Each cycle is 28 days. Each cohort can enroll 19 patients, with a decision to stop or proceed to 54 patients. The primary objective of this study is to assess whether BP1001 + decitabine provides higher complete remission rates than decitabine alone (by historical comparison) in AML/high risk MDS patients. The secondary objectives of this study are to assess: Safety of BP1001 + decitabine; partial remissions and blast count reductions; overall survival, time to response and duration of response; minimal residual disease status in patients who achieve complete remission; and plasma PK profile of BP1001. The exploratory objective of this study is to evaluate the association of treatment response with cytogenetic and molecular characteristics. Clinical trial information: NCT02781883 .

Antisense oligonucleotides, siRNA, anti-microRNA are designed to selectively bind to target mRNAs, and silence disease-causing or -associated proteins. The clinical development of nucleic acid drugs has been limited by their poor bioavailability.This review article examines the strategies that have been utilized to improve the bioavailability of nucleic acids. The chemical modifications made to nucleic acids that have improved their resistance against nuclease degradation are briefly discussed. The design of cationic and neutral lipid nanoparticles that enable the systemic delivery of nucleic acids in vivo is reviewed, and the proof-of-concept evidence that intravenous administration of nucleic acids incorporated into lipid nanoparticles leads to decreased expression of target genes in humans. Preclinical results of the neutral BP-100-1.01 nanoparticle are highlighted.To further improve the clinical potential of nucleic acid cancer drugs, we predict research on the next generation of lipid nanoparticles will focus on: i) enhancing nucleic acid delivery to poorly vascularized tumors, as well as tumors behind the blood-brain barrier; and ii) improving the accessibility of nucleic acids to the cytoplasm by enhancing endosomal escape of nucleic acids and/or reducing exocytosis of nucleic acids to the external milieu.

Summary Background Activating mutations of tyrosine kinases are common in leukaemias. Oncogenic tyrosine kinases use the growth factor receptor-bound protein 2 (Grb2) for signal transduction, leading to activation of mitogen-activated protein kinase (MAPK) 1 and MAPK3 (ERK2 and ERK1). We hypothesised that inhibition of Grb2 would suppress ERK1 and ERK2 activation and inhibit leukaemia progression. To inhibit Grb2, a liposome-incorporated antisense oligodeoxynucleotide that blocks Grb2 protein expression, BP1001, was developed. We report the first phase 1 findings of BP1001. Methods In this single-centre, open-label, dose-escalation phase 1/1b trial, we enrolled participants (aged ≥18 years) with refractory or relapsed acute myeloid leukaemia, Philadelphia-chromosome-positive chronic myeloid leukaemia (in chronic, accelerated, or blast phase), acute lymphoblastic leukaemia, or myelodysplastic syndrome, at MD Anderson Cancer Center (Houston, TX, USA). We used a 3 + 3 dose escalation strategy, with at least three patients enrolled at each dose level. We administered BP1001 intravenously, twice weekly, for 28 days, with a starting dose of 5 mg/m 2 . If two or more patients developed toxic effects of grade 3 or higher, that dose level was deemed toxic. The dose was escalated if it did not produce dose-limiting toxic effects, and patients would be sequentially enrolled into cohort 2 (10 mg/m 2 ), cohort 3 (20 mg/m 2 ), cohort 4 (40 mg/m 2 ), cohort 5 (60 mg/m 2 ), or cohort 6 (90 mg/m 2 ). After completion of monotherapy, we assessed the safety and toxicity of BP1001 (60 or 90 mg/m 2 ) in combination with 20 mg low-dose cytarabine (twice-daily subcutaneous injections) in a phase 1b study in patients with refractory or relapsed acute myeloid leukaemia (ie, those who were refractory to at least one previous therapy regimen and no more than one previous salvage regimen). The objectives of this study were to establish the toxicity and tolerance of escalating doses of BP1001 monotherapy in patients with refractory or relapsed leukaemia, to assess the maximum tolerated dose of BP1001, and to determine the optimal biologically active dose of BP1001, defined as a 50% reduction in Grb2 expression in circulating leukaemia cells. We also aimed to assess the in-vivo pharmacokinetics of BP1001 and tumour response. The study is completed and is registered with ClinicalTrials.gov, number NCT01159028. Findings Between July 23, 2010, and Feb 23, 2016, we enrolled and treated 39 patients, of whom 27 were assessable for dose-limiting toxicity. The first patient treated had mucositis and hand–foot syndrome, which were assessed as possibly related to BP1001 and counted as a dose-limiting toxicity. We noted no other dose-limiting toxicities, and a maximum tolerated dose was not identified. The highest tested dose of BP1001 was 90 mg/m 2 . The most common grade 3–4 adverse events were cardiopulmonary disorders (25 [64%] of 39 patients), and fever (including neutropenic fever) and infections (17 [44%] patients). Grade 5 adverse events were cardiopulmonary disorders (two [5%] of 39 patients), fever (including neutropenic fever) and infections (two [5%] of 39 patients), and multi-organ failure (one [3%] of 39 patients). Nine (33%) of 27 patients who had peripheral blood blasts at the start of therapy had a reduction of 50% or more in peripheral blood blasts while receiving BP1001 montherapy. Three (10%) of 29 patients who had bone marrow blasts at the start of therapy had a reduction in bone marrow blasts of 50% or more while receiving BP1001 monotherapy. Per investigator's assessment, seven (22%) of 32 patients benefited from BP1001 monotherapy and had extended cycles of treatment. Of seven patients receiving BP1001 plus low-dose cytarabine combination therapy, two had complete remission, one had complete remission with incomplete haematological recovery, and two had stable disease with no dose-limiting toxicity; one patient died and one withdrew, both because of disease progression. There were eight deaths; none were treatment related. Interpretation BP1001 is well tolerated, with early evidence of anti-leukaemic activity in combination with low-dose cytarabine. To further explore this anti-leukaemic activity, the efficacy of BP1001 plus low-dose cytarabine combination is being investigated in an ongoing phase 2 study in patients with previously untreated acute myeloid leukaemia who are ineligible for intensive induction therapy. Funding Bio-Path Holdings Inc.

Background: Signal Transduction and Activator of Transcription-3 (STAT3), though typically inactive in normal cells, is aberrantly active in cancer cells. Activation of STAT3 has been found in many types of cancers, including non-small cell lung cancer (NSCLC), acute myelogenous leukemia (AML), and pancreatic ductal adenocarcinoma (PDAC). Activation of STAT3 correlates with poor clinical outcome, high grade disease and metastasis, and has been linked with resistance to chemotherapy, including gemcitabine, which is one of the standard of care agents for advanced PDAC. Therefore, inhibition of STAT3 in combination with chemotherapy is expected to produce clinical benefit. We have developed BP1003, a novel liposome-incorporated STAT3 antisense oligodeoxynucleotide (oligo) as a specific inhibitor of STAT3. Methods: Four candidate antisense oligo sequences directed against STAT3 mRNA were initially identified. They were manufactured as nuclease-resistant P-ethoxy oligos and incorporated into neutral dioleoylphosphatidylcholine liposomes. Cell viability tests and Western blots were conducted to determine the inhibitory effects of liposome-incorporated STAT3 antisense oligo on NSCLC and AML cells. Ex vivo live tissue sensitivity assay (LTSA) was performed with a panel of 20 PDAC patients-derived xenografts (PDXs) to study the overall activity of BP1003, alone and in combination with gemcitabine. Using previous defined criteria, tissue slice viability inhibition greater than 30% and p Results: The most potent liposome-incorporated STAT3 antisense sequence in decreasing NSCLC cell viability was selected as the candidate BP1003 drug. Further validation in AML cells demonstrated that BP1003 inhibited cell viability and STAT3 protein expression. In the ex vivo LTSA assay, BP1003, at a dose of 10 µM, significantly inhibited the tissue slice viability of 9 out of 18 PDAC PDXs by over 30% (p Conclusions: Liposome-incorporated STAT3 antisense oligo, BP1003, shows promising in vitro and in vivo preclinical activity as single and combination agent and might be a novel therapeutic strategy in patients with advanced tumors and/or metastasis. Citation Format: Bingbing Dai, Ana Tari Ashizawa, Jithesh J. Augustine, Michael Kim, Jason Fleming. BP1003, a novel liposome-incorporated STAT3 antisense oligodeoxynucleotide inhibitor [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 4786.

Background: The growth factor receptor bound protein-2 (Grb2) is vital to tumor progression. Prexigebersen (BP1001) is a liposome-incorporated Grb2 antisense oligonucleotide that inhibits Grb2 expression. Grb2 inhibition suppresses cancer growth and survival. A Phase I/IB single center study (Ohanian, Lancet Haematol 2018) in relapsed/refractory AML patients (pts) demonstrated BP1001 safety up to 90 mg/m2 and demonstrated CR/CRi in 3/6 relapsed/refractory AML pts treated with BP1001 + low dose cytarabine (LDAC) in the Phase IB portion. Based on PK considerations, the recommended Phase II dose was 60 mg/m2. A phase II study was initiated to explore the clinical impact of BP1001 in untreated AML pts considered unsuitable for standard chemotherapy. Methods: This is a multi-center open-label, Phase II study in untreated AML pts to determine the safety and preliminary efficacy of BP1001 (given at 60 mg/m2 over 1-3 h twice weekly for a total of 8 doses over a 28-day cycle) in combination with LDAC (starting after the Day 4 dose, 20 mg twice daily for 10 days). Eligible pts were considered unsuitable for or refused intensive chemotherapy and had ECOG performance status of 0-2. Blood samples were taken on Days 1 and 11 to determine BP1001 plasma pharmacokinetics (PK). Results: A total of 25 pts (16 male: 64%) with a median age of 74 years (range, 51-86 years) was treated with at least 1 cycle of BP1001 + LDAC. Sixty-eight percent (17/25) had secondary AML evolved from MDS (n=13) or MPN (n=3) or prior chemotherapy/radiotherapy (n=1). The majority (64%) had adverse-risk (ELN classification); 36% were intermediate-risk. Infusional reactions (IR) (rigors, fevers, or chills) were observed in 20% of pts when the infusion rate was 1 h. IR were mild, controlled with supportive measures, and were not observed when the infusion rate was 2 h. Other AE's were generally consistent with those expected with LDAC and/or AML, including fatigue (64%), diarrhea (24%), neutropenic fever (24%), and pneumonia (20%). The most frequent SAEs were pneumonia (20%) and neutropenic fever (24%). Four pts died within 30 days of treatment (between days 20-27), all of AML-related complications (including intracranial hemorrhage, pneumonia, septic shock, and splenic rupture). Ten pts had documented progressive disease. Two pts withdrew consent after 1 cycle of treatment. Evaluability of efficacy was defined as: receipt of at least 4 cycles of therapy, PD, or CR/CRi prior to 4 cycles. PK data revealed that the Time of maximal observed concentration (Tmax), the mean terminal elimination half-life (t1/2), and the area under the curve from zero to the last quantifiable concentration (AUC(last)) for BP1001 were similar between Days 1 and 11. Tmax of BP1001 was 1.9 vs 2.3 h (day 1 vs 11); t1/2 was 5.2 vs 5.7 h; AUC(last) was 308 vs 275 h*ng/mL. Response: Among 17 fully evaluable pts, 7 pts (41%) demonstrated bone marrow blast reduction, including 5 (29%) who achieved a CR (n=3), CRi (n=1), or morphologic leukemia free state (n=1). Two additional pts demonstrated a ≥50% reduction of bone marrow blasts (from 20% to 9% and from 71% to 8%), including 1 with hematologic improvement of the absolute neutrophil count. The median overall survival (OS) was 5.2 months (range, 0.6-15.2) for all patients. For evaluable patients, the median OS was 6.3 months (0.8-15.2 months). The CRs included: a 74-year old female who achieved CR after 2 cycles of therapy and then received 2 additional cycles followed by a matched-sibling allogeneic transplant. She remained in CR for 10 months before first relapse. A 73-year old female, with AML transformed from MDS, benefited from therapy despite refusal of blood products due to religious objections. Her hemoglobin declined to 4.0 g/dL during therapy but recovered to as high as 9.9 g/dL once in CR after 4 cycles. An 86-year old male achieved CR after 4 cycles of therapy and maintained CR for 3 months before relapse. Conclusions: BP1001 has been safely administered to 25 pts with untreated AML, who were considered unsuitable for standard chemotherapy. Efficacy data are encouraging in a challenging population in which the majority of pts had secondary AML or adverse-risk AML, and compares favorably to the reported CR/CRi/CRp rate with LDAC of 7-13% (Heiblig, Mediterr J Hematol 2016; Kantarjian, J Clin Oncol 2012; Döhner, Blood 2014). A protocol amendment made to optimize BP1001 administration with LDAC and to add a decitabine combination arm is in place. Disclosures Lin: Jazz Pharmaceuticals: Honoraria. Ritchie:Incyte: Consultancy, Speakers Bureau; Astellas Pharma: Research Funding; NS Pharma: Research Funding; Novartis: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding, Speakers Bureau; ARIAD Pharmaceuticals: Speakers Bureau; Bristol-Myers Squibb: Research Funding; Pfizer: Consultancy, Research Funding; Celgene: Consultancy, Other: Travel, Accommodations, Expenses, Speakers Bureau. Craig:Celgene: Research Funding; Novartis: Research Funding; Actinium Pharmaceuticals: Research Funding. Agrawal:Incyte: Speakers Bureau. Pemmaraju:Affymetrix: Research Funding; plexxikon: Research Funding; daiichi sankyo: Research Funding; SagerStrong Foundation: Research Funding; samus: Research Funding; celgene: Consultancy, Honoraria; abbvie: Research Funding; cellectis: Research Funding; stemline: Consultancy, Honoraria, Research Funding; novartis: Research Funding. Tari Ashizawa:Bio-Path Holdings, Inc.: Employment, Equity Ownership, Patents & Royalties. Hahne:Bio-Path Holdings, Inc.: Employment. Cortes:Daiichi Sankyo: Consultancy, Research Funding; Astellas Pharma: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Arog: Research Funding. Roboz:Orsenix: Consultancy; Astex Pharmaceuticals: Consultancy; Argenx: Consultancy; Cellectis: Research Funding; Roche/Genentech: Consultancy; Novartis: Consultancy; Janssen Pharmaceuticals: Consultancy; Aphivena Therapeutics: Consultancy; Celltrion: Consultancy; Otsuka: Consultancy; Sandoz: Consultancy; Orsenix: Consultancy; Roche/Genentech: Consultancy; Celgene Corporation: Consultancy; Astex Pharmaceuticals: Consultancy; AbbVie: Consultancy; Daiichi Sankyo: Consultancy; Cellectis: Research Funding; Aphivena Therapeutics: Consultancy; Janssen Pharmaceuticals: Consultancy; Novartis: Consultancy; Otsuka: Consultancy; Pfizer: Consultancy; Jazz Pharmaceuticals: Consultancy; Eisai: Consultancy; Eisai: Consultancy; Argenx: Consultancy; Sandoz: Consultancy; Pfizer: Consultancy; Celgene Corporation: Consultancy; Daiichi Sankyo: Consultancy; AbbVie: Consultancy; Bayer: Consultancy; Celltrion: Consultancy; Jazz Pharmaceuticals: Consultancy; Bayer: Consultancy.

Prexigebersen (liposome-incorporated nuclease resistant antisense oligonucleotides specific for GRB2, L-GRB2) has proven highly effective in the treatment of leukemia, however its application in solid malignancies including gynecologic cancer has yet to be investigated. Given the biological significance of GRB2, we aim to assess the effect of prexigebersen in pre-clinical models. We tested the clinical significance of GRB2 using publicly available high-throughput data. We investigated the expression of GRB2 in a series of in vitro (Western Blotting) experiments in high-grade serous (HGSC) and uterine (UC) carcinoma models. We also tested in vivo (orthotopic mouse model) the biological effects of prexigebersen in HGSC models (OVCAR 5), first in a dose-defining experiment then in combination with standard dose paclitaxel. We first examined GRB2 using TCGA and GISTIC data and found alterations (amplifications, deletions and mutations) in 6% (33 of 594) of HGSC cases and 5% (25 of 547) of UC cases. Amplifications of GRB2 are associated with a decreased patient survival (p Citation Format: Olivia D. Lara, Cristian Rodriquez-Aguayo, Emine Bayrakter, Paola Amero, Selanere Mangala, Chris LaFargue, Ana Tari-Ashizawa, Gabriel Lopez-Berestein, Anil Sood. Grabbing GRB2: The use of liposome-incorporated Grb2 antisense oligonucleotides as a novel therapy in gynecologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5786.

A major obstacle in the treatment of glioblastoma (GBM) is the blood brain barrier (BBB), which restricts drug molecules in the bloodstream from reaching the tumor. The BBB may be disrupted at or near the GBM tumor core, but it is intact at the growing edge of the tumor, where invasive cells reside and can cause disease recurrence. To combat GBM more effectively, novel drug transport technology that crosses intact BBB is urgently needed. Brain microvascular endothelial cells (BMEC) found in the BBB are lined with low-density lipoprotein receptors (LDLr) and LDL-like receptor proteins (LRP), which are essential to the endocytosis/ transcytosis processes of BMEC. GBM cells are known to express higher levels of LDLr and LRP than normal brain cells. The objective of our research is to develop a novel lipid nanoparticle (NP) that targets LDLr and LRP to facilitate BMEC endocytosis/ transcytosis and GBM uptake. We focused on developing antisense oligonucleotides (oligos) as the drug cargo for our NP. Less than 20% of disease-associated genes are targetable by conventional drug molecules; however, we hypothesize that disease-associated genes, like EZH2 and ZEB1 transcriptional repressors, are amendable to antisense oligo inhibition. We engineered a novel NP which is composed of neutral lipids: phosphatidylcholine (PC), cholesterol (Chol) and a polymer called poloxamer (PXR). Toxicology studies indicated that NP composed of PC/Chol/PXR did not have adverse effects on mice. Flow cytometry and fluorescent microscopy revealed that PC/Chol/PXR NP was avidly taken up by BMEC, and that PC/Chol/PXR efficiently delivered antisense oligos to GBM cells. Additionally, intravenous injection of PC/Chol/PXR led to deposition of antisense oligos in intracranial GBM xenografts. Future studies will determine the efficacy of the innovative PC/Chol/PXR NP in suppressing target gene expression in intracranial GBM. (Supported by Florida Center for Brain Tumor Research and Accelerate Brain Cancer Cure).

Background: The Growth factor receptor bound protein-2 (Grb2) is essential to cancer cell signaling. Grb2 binds to numerous tyrosine kinases, including the Bcr-Abl chimeric oncogene found in Philadelphia chromosome positive chronic myelogenous leukemia (CML) cells. Upon binding to tyrosine kinases, Grb2 transduces the kinases signals to the activation of Ras and its downstream protein, Extracellular signals regulated protein kinases-1,2 (Erk1,2). The direct binding of Grb2 to Bcr-Abl is required for Bcr-Abl to transform fibroblasts and induce CML-like diseases in mice. We hypothesize that Grb2 is a potential therapeutic target against CML. BP1001, a liposome-incorporated antisense oligodeoxynucleotide targeted against the Grb2 mRNA, was developed to inhibit Grb2 expression. Previously we demonstrated that BP1001 suppressed the growth of CML cell lines and CML xenografts. Aim: Since the clinical activity of BP1001 will be investigated in gleevec-resistant CML patients, preclinical studies were conducted to determine whether BP1001 could suppress the proliferation of gleevec-resistant CML cells. Additionally, whether BP1001 could enhance the inhibitory effects of dasatinib in CML cells was determined. The clinical activity of BP1001 was studied in CML blast patients, who were part of a larger Phase I clinical study. Methods: The alamarBlue dye incorporation method and the CellTiter Glow method were used to determine cell viability. Flow cytometry was utilized to analyze cell cycling and Grb2 expression. The clinical study was a standard 3+3 dose-finding study in which BP1001 was given twice weekly, IV over 2-3 hours for 28 days. The clinical activity of BP1001 was evaluated in its ability to reduce target Grb2 protein in CD33-expressing cells and to reduce leukemia blast counts. Results: BP1001 decreased the proliferation of gleevec-resistant CML cells in a dose-dependent manner. Furthermore, BP1001 pretreatment enhanced the inhibitory effects of dasatinib in CML cells, by about 2-6 fold. The major inhibition mechanism of the BP1001 + dasatinib combination was cell death. A total of39 patients with refractory/relapsed hematologic malignancies was enrolled in the clinical trial. Five were CML blast phase patients; they were enrolled in the first cohort (5 mg/m2BP1001). Two CML patients, who had T315I mutation, showed significant reductions in circulating blasts during treatment. One patient's blasts were reduced from 89% to 9%, while another patient's blasts were reduced from 17% to 8%. BP1001 decreased >25% Grb2 levels in 10 of 12 samples (average decrease: 49%) and >25% phosphorylated Erk1,2 levels in 7 of 12 samples (average decrease: 52%). Conclusions: BP1001 was active against gleevec-resistant CML cells and enhanced dasatinib efficacy in preclinical studies. BP1001 may have therapeutic activity against CML. We are planning a Phase I/II trial to study the safety and efficacy of BP1001 in combination with dasatinib in accelerated or blast phase CML patients. Disclosures Tari Ashizawa: Bio-Path Holdings, Inc.: Employment, Equity Ownership.

While a wealth of literature for tissue-specific liposomes is emerging, optimal formulations to target the cells of the peripheral nervous system (PNS) are lacking. In this study, we asked whether a novel formulation of phospholipid-based liposomes could be optimized for preferential uptake by microvascular endothelia, peripheral neurons and Schwann cells. Here, we report a unique formulation consisting of a phospholipid, a polymer surfactant and cholesterol that result in enhanced uptake by targeted cells. Using fluorescently labeled liposomes, we followed particle internalization and trafficking through a distinct route from dextran and escape from degradative compartments, such as lysosomes. In cultures of non-myelinating Schwann cells, liposomes associate with the lipid raft marker Cholera toxin, and their internalization is inhibited by disruption of lipid rafts or actin polymerization. In contrast, pharmacological inhibition of clathrin-mediated endocytosis does not significantly impact liposome entry. To evaluate the efficacy of liposome targeting in tissues, we utilized myelinating explant cultures of dorsal root ganglia and isolated diaphragm preparations, both of which contain peripheral neurons and myelinating Schwann cells. In these models, we detected preferential liposome uptake into neurons and glial cells in comparison to surrounding muscle tissue. Furthermore, in vivo liposome administration by intramuscular or intravenous injection confirmed that the particles were delivered to myelinated peripheral nerves. Within the CNS, we detected the liposomes in choroid epithelium, but not in myelinated white matter regions or in brain parenchyma. The described nanoparticles represent a novel neurophilic delivery vehicle for targeting small therapeutic compounds, biological molecules, or imaging reagents into peripheral neurons and Schwann cells, and provide a major advancement toward developing effective therapies for peripheral neuropathies.

7010Background: Essential to cancer cell signaling, the Growth factor Receptor Bound protein-2 (Grb2) is utilized by oncogenic tyrosine kinases to activate Ras and ERK. BP1001is a liposome-incorpor...

Glioblastoma (GBM) remains one of the most lethal types of cancer. Novel therapies are urgently needed for patients with GBM. Recently we demonstrated high expression of ZEB-1 but low expression of microRNA-200 (miR-200) in GBM samples, and that suppressing ZEB-1 expression or re-expressing miR-200 in GBM cells decreased GBM invasion, increased sensitivity to the chemotherapy temozolomide, and prolonged survival in mouse xenotransplantation studies. Therefore, using nucleic acids to modify ZEB-1 or miR-200 expression is a potential novel GBM therapeutic strategy. However, the transport technology that enables nucleic acids to cross the blood brain barrier (BBB) and enter GBM is not available. Brain microvascular endothelial cells (BMEC) found in the BBB are lined with caveolin, scavenger receptors class B type I (SR-B1), and low-density lipoprotein receptors (LDLr), which are essential to the endocytosis/ transcytosis processes of BMEC. We engineered a novel lipid nanoparticle (LNP) that targets caveolin, SR-BI, and LDLr on BMEC. The LNP is composed of phosphatidylcholine (PC), cholesterol (Chol) and poloxamer (PXR). Flow cytometric analysis indicates that LNP composed of PC/Chol/PXR are avidly taken up by BMEC, approximately 23-fold higher than LNP composed of PC only. Intravenous injection of PC/Chol/PXR, but not PC, LNP in mice leads to their accumulation in the central nervous system including the hippocampus and the cerebellum. Fluorescent microscopy reveals that PC/Chol/PXR LNP efficiently delivers fluorescent nucleic acids to human GBM cells, which express 2- to 5-fold higher levels of caveolin than normal brain cells. Our data indicate that the innovative PC/Chol/PXR LNP has high potential to deliver nucleic acids to GBM in vivo, and enable the utility of nucleic acids as a therapeutic modality against GBM. (Supported by UF Research Opportunity Seed Fund, McKnight Brain Institute Therapeutic Discovery and Translational Research Award, NIH/NINDS NS055165 and the McKinney, Maren, and Thompson Regenerative Medicine Funds).