SGN-CD33A in Combination with Cytarabine or Hypomethylating Agents Demonstrates Enhanced Anti-Leukemic Activity in Preclinical Models of AML

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3739-3739 ◽  
Author(s):  
May S.K. Sutherland ◽  
Changpu Yu ◽  
Martha Anderson ◽  
Kim Emmerton ◽  
Weiping Zeng ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Tumor Growth ◽  
Cell Lines ◽  
Low Dose ◽  
Drug Resistant ◽  
Hypomethylating Agents ◽  
Preclinical Models ◽  
Employment Equity ◽  
Myeloid Malignancies ◽  
Equity Ownership

Abstract Long-term survival rates for acute myeloid leukemia (AML) patients remain poor, highlighting the need for further treatment options. AML cells express the myeloid marker CD33, making them amenable to CD33-targeted therapy. SGN-CD33A is a novel anti-CD33 antibody-drug conjugate (ADC) composed of a humanized antibody conjugated to a highly potent DNA-binding pyrrolobenzodiazepine (PBD) dimer drug via a protease-cleavable dipeptide linker. An engineered cysteine on each heavy chain attaching the PBD dimer to the antibody allows uniform drug loading of approximately two PBD dimers per antibody. Upon binding to CD33 on the cell surface, SGN-CD33A is internalized, the linker is cleaved by proteases in the lysosomes, and the released drug forms DNA crosslinks, resulting in cell death. SGN-CD33A is active as a single agent against a broad panel of primary AML samples and in preclinical models of AML that are characteristically resistant to chemotherapy (multi-drug-resistant, MDR-positive) (Sutherland et al. Blood 2013). In the present study, we tested the activity of SGN-CD33A in cytotoxicity assays in combination with therapies commonly used in the treatment of myeloid malignancies including cytarabine (Ara-C) and the hypomethylating agents, 5-azacytidine (vidaza) or 5-aza-2-deoxcytidine (decitabine). Significant synergism in tumor cell killing, as assessed by the Chou-Talalaly Combination Index (CI), was observed when MDR-positive AML cell lines, KG-1 and TF1-a, were treated simultaneously with the combination of SGN-CD33A and Ara-C (CI < 0.7) or SGN-CD33A and a hypomethylating agent (CI < 0.7). Consistent with these observations, mouse xenograft experiments were conducted with AML cell lines, and demonstrated improved antitumor activity with the combinations compared to either agent alone. In the subcutaneous MDR-positive TF1-a model of AML, a single low dose of SGN-CD33A (30 mcg/kg) in combination with Ara-C significantly reduced tumor burden compared to either agent alone or to the nonbinding control ADC groups (p< 0.0001). Significant reductions in tumor growth were also observed in subcutaneous MDR-positive TF1-a or KG-1 murine models of AML treated with SGN-CD33A in combination with hypomethylating agents. Whereas a single low dose of SGN-CD33A or the hypomethylating agents decitabine or vidaza delayed tumor growth, the combination delivered greater antitumor activity than the individual agents alone. These findings demonstrate that SGN-CD33A can be combined with therapies that are commonly used in treating myeloid malignancies to deliver significantly improved antitumor activity in preclinical drug-resistant models of AML. Disclosures Sutherland: Seattle Genetics: Employment, Equity Ownership. Yu:Seattle Genetics: Employment, Equity Ownership. Anderson:Seattle Genetics: Employment, Equity Ownership. Emmerton:Seattle Genetics: Employment, Equity Ownership. Zeng:Seattle Genetics: Employment, Equity Ownership. O'Meara:Seattle Genetics, Inc.: Employment, Equity Ownership. Kennedy:Seattle Genetics, Inc.: Employment, Equity Ownership. Ryan:Seattle Genetics: Employment, Equity Ownership. Benjamin:Seattle Genetics: Employment, Equity Ownership.

scholarly journals SGN-CD33A in Combination with Hypomethylating Agents Is Highly Efficacious in Preclinical Models of AML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3785-3785 ◽  
Author(s):  
May S.K. Sutherland ◽  
Changpu Yu ◽  
Christine O'Day ◽  
Steve Alley ◽  
Martha Anderson ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Antitumor Activity ◽  
Tumor Growth ◽  
Cell Lines ◽  
Drug Resistant ◽  
Hypomethylating Agents ◽  
Preclinical Models ◽  
Employment Equity ◽  
Equity Ownership

Abstract Acute myeloid leukemia (AML) remains a therapeutic challenge. Long-term survival rates, in particular for older AML patients, remain poor, highlighting the need for improved and well-tolerated treatment options. AML patients who are unfit for high dose chemotherapy in the US are often prescribed hypomethylating agents (azacitidine or decitabine) although the efficacy of these agents in this population was modest (Estey, Leukemia 2013). SGN-CD33A is a CD33-directed antibody drug conjugate (ADC) that is comprised of a cysteine-engineered anti-CD33 antibody, a cleavable dipeptide linker that is highly stable in circulation, and a PBD dimer that binds DNA with high intrinsic affinity. The ADC is active as a single agent in preclinical models of AML that are characteristically resistant to chemotherapy (multi-drug-resistant, MDR-positive) (Sutherland et al. Blood 2013). In the present study, we tested the activity of SGN-CD33A in combination with hypomethylating agents (HMA), azacitidine or decitabine. We hypothesized that the combination of SGN-CD33A with an HMA will have greater impact on the DNA repair pathway in leukemic cells, furthering the processes of apoptosis and cell death. MDR-positive AML cells were treated for 96 hours with SGN-CD33A and each of the HMAs alone and in combination, evaluating both simultaneous as well as sequential administration. Enhanced tumor cell killing was observed when AML cells were treated concomitantly with the combination of SGN-CD33A and an HMA or pre-treated with HMAs prior to the addition of SGN-CD33A. Superior anti-leukemic activity of subtherapeutic doses of SGN-CD33A in combination with HMAs was observed in mouse xenograft models generated with MDR-positive AML cell lines. In the difficult to treat HEL9217 model, decitabine or a single dose of 200 mcg/kg SGN-CD33A delayed tumor growth, while significant reductions in tumor growth were observed for the combination treatment (p=0.0001). Improved antitumor activity in this model was also observed for SGN-CD33A in combination with azacitidine. SGN-CD33A in combination with decitabine significantly reduced tumor burden compared to either agent alone in the TF1-α AML model (p=0.0002). Similarly, sequenced dosing of azacitidine followed by a subtherapeutic dose of 30 mcg/kg SGN-CD33A in mice bearing KG-1 xenografts delivered greater antitumor activity compared to the individual agents (p=0.0001). To investigate the mechanism underlying the enhancement in antileukemic activity, we looked at the impact of HMAs alone and in combination with SGN-CD33A on myeloid marker expression, PBD drug release, and impact on the DNA damage and apoptotic pathways. In AML cell lines that did not show improved cytotoxic activity with the combination of SGN-CD33A and HMA, HMA treatment appeared to have no positive effect on the cell surface levels of CD33. However in responsive cell lines such as TF1-α, HMA treatment resulted in time- and dose-dependent increases in the levels of CD33. Significant increases in expression were observed between 2 and 4 days with decitabine and after 4 days with azacitidine. In addition, more PBD dimer drug from SGN-CD33A was incorporated into DNA in HMA treated cells. Concomitant, the treatment of AML cells with the combination of SGN-CD33A and HMA resulted in greater DNA damage and apoptosis as shown by the increased levels of the early DNA damage marker, γH2AX, and the formation of cleaved PARP, an apoptosis marker, compared to either agent alone. Studies are in progress to investigate the effects on other components of the DNA repair and cell cycle pathways. These findings demonstrate that SGN-CD33A can be successfully combined with hypomethylating agents to deliver marked antitumor activity in preclinical drug-resistant models of AML. Disclosures Sutherland: Seattle Genetics, Inc.: Employment. Yu:Seattle Genetics, Inc.: Employment. O'Day:Seattle Genetics,Inc: Employment. Alley:Seattle Genetics,Inc.: Employment. Anderson:Seattle Genetics, Inc.: Employment. Emmerton:Seattle Genetics, Inc.: Employment. Zeng:Seattle Genetics, Inc.: Employment. O'Meara:Seattle Genetics, Inc: Employment, Equity Ownership. Feldman:Seattle Genetics,Inc: Employment. Kennedy:Seattle Genetics,Inc: Employment, Equity Ownership, Honoraria, Speakers Bureau. Ryan:Seattle Genetics, Inc.: Employment. Benjamin:Seattle Genetics, Inc: Employment.

scholarly journals Clinical Pharmacodynamic Markers and Combinations with SY1425 (tamibarotene) in a Genomically-Defined Subset of Non-APL AML

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2898-2898
Author(s):  
Michael R McKeown ◽  
Christopher Fiore ◽  
Emily Lee ◽  
Matthew L Eaton ◽  
Christian C. Fritz
Keyword(s):  
Transcription Factor ◽  
Cell Lines ◽  
Binding Sites ◽  
Clinical Studies ◽  
High Cell ◽  
Preclinical Models ◽  
Employment Equity ◽  
Equity Ownership ◽  
Maximal Induction

Abstract SY-1425, a potent and selective agonist of the retinoic acid receptor RARα, is being investigated in a Ph2 trial in a novel genomically-defined subset of non-APL AML and MDS patients (clinicaltrials.gov NCT02807558). RARa is a nuclear hormone receptor and transcription factor that regulates genes involved in cell differentiation and proliferation. We identified a super-enhancer (SE) at the RARA locus, the gene encoding RARa, in a subset of primary non-APL AML blasts. Preclinical models demonstrated a correlation between the presence of a RARA SE and sensitivity to SY-1425, providing the rationale for clinical investigation. Further research has investigated pharmacodynamics (PD) markers and combinations of drugs to support clinical development of SY-1425. In this study we identified DHRS3mRNA induction as a measure of RARα target engagement with SY-1425. We also demonstrated synergy in preclinical models with SY-1425 and hypomethylating agents. Since RARα is a transcription factor that regulates target genes when bound by a retinoid, we characterized the dynamic expression changes of a panel of RARA enhancer- high and - low non-APL AML cell lines (hereafter referred to as RARA-high and -low) in response to SY-1425 treatment. DHRS3 showed the largest expression increase following treatment in 3 RARA-high cell lines, with a range of 29 to 115 fold. In contrast, there was a much lower DHRS3 induction in 3 RARA-low cell lines (range of 1.6 to 6.1 fold). Induction was found to be both time- and dose-dependent with maximal induction at approximately 6 hours and half maximal induction near the EC50 for the anti-proliferative effect in RARA-high cell lines. DHRS3 encodes dehydrogenase/reductase (SDR family) member 3, a metabolic enzyme involved in maintaining cellular retinol homeostasis and had previously been shown to be induced by retinoids. Thus, DHRS3induction in tumor cells represents a potentially useful PD marker for clinical studies of SY-1425. To better understand the mechanism of induction of DHRS3 by SY-1425 we examined the chromosomal localization of RARα as well as the epigenomic state of the DHRS3 locus by ChIP-seq for RARα and H3K27 acetylation, the latter being an indicator of active enhancers and promoters. In the untreated state, OCI-AML3 (a typical RARA-high AML cell line) was found to have multiple RARα binding sites both within and distal to the DHRS3 gene but minimal H3K27 acetylation. Following treatment with SY-1425, the level of H3K27 acetylation at DHRS3 increased, resulting in the formation of a SE. Moreover, the SE encompassed the RARα binding sites, consistent with the model in which SY-1425 converts RARα into an activator of DHRS3expression. Similar results were seen for the CD38 locus in which SY-1425 treatment increased expression, H3K27 acetylation, and RARα binding. CD38 is a cell surface antigen and marker of myeloid maturation readily analyzed by FACS analysis, suggesting it could be an additional PD marker to be used in clinical studies. Indeed, it was found that SY-1425 induced CD38 cell surface expression at similar levels in RARA-high AML cell lines and the NB-4 APL cell line, but not in RARA-low cell lines. We also investigated combinations of SY-1425 with approved or investigational AML and MDS agents in in vitro and in vivo models to inform future clinical studies and to further explore potential PD markers unique to the combined action of the drugs. Several standard of care agents and drugs in current development were found to have synergistic interactions with SY-1425 in RARA-high but not RARA-low cell lines. In particular, azacitidine and decitabine each showed strong in vitro synergy with SY-1425. Evaluation of SY-1425 plus azacitidine in a RARA-high PDX model of non-APL AML demonstrated a better response compared to either agent alone. Additional genome-wide ChIP-seq and expression studies of RARA-high cells treated with various combinations are being investigated to identify optimal PD markers for these combinations. These studies support the use of DHRS3 mRNA induction in tumor cells as a PD marker in the recently initiated Ph2 study of SY-1425 in genomically-defined non-APL AML and MDS patients (clinicaltrials.gov NCT02807558) and further exploration as a PD marker for future combination studies. Disclosures McKeown: Syros Pharmaceuticals: Employment, Equity Ownership. Fiore:Syros Pharmaceuticals: Employment, Equity Ownership. Lee:Syros Pharmaceuticals: Employment, Equity Ownership. Eaton:Syros Pharmaceuticals: Employment, Equity Ownership. Fritz:Syros Pharmaceuticals: Employment, Equity Ownership.

The Novel mTOR Kinase Inhibitor CC-223 Demonstrates Significant Activity In In Vitro Models Of Multiple Myeloma (MM), Both As a Single Agent and In Combination With The Approved Agents, Dexamethasone, Lenalidomide and Pomalidomide

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3160-3160 ◽  
Author(s):  
Emily Rychak ◽  
Derek Mendy ◽  
Karen Miller ◽  
Jim Leisten ◽  
Rama Krishna Narla ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Death ◽  
Tumor Growth ◽  
Cell Lines ◽  
Combination Index ◽  
Synergistic Effects ◽  
Standard Of Care ◽  
Employment Equity ◽  
Equity Ownership

Abstract Over expression of the PI3 kinase/mTOR/AKT pathway has been well documented in MM patient biopsies and human MM cell lines, suggesting this pathway plays a key role in the survival and proliferation of malignant plasma cells. Rapamycin and the rapalogs are allosteric inhibitors of the mTORC1 complex (consisting of mTOR, raptor, mLST8 and PRAS40), inducing mainly cytostatic effects but not cell death. Inhibition of mTORC1 prevents a negative feedback loop to the mTORC2 complex (consisting of mTOR, Rictor, mLST8 and Sin 1) leading to the phosphorylation of AKT. Phosphorylated AKT is a key inducer of anti-apoptosis mechanisms and cell cycle progression, which may explain the limited results of the rapalogs in the clinic. Recently developed mTOR kinase inhibitors (i.e., CC-223) target both mTORC1 and mTORC2 complexes in order to inhibit tumor growth and importantly, induce cell death. Here we evaluate the effects of CC-223 on a panel of MM cell lines, in combination with current standard of care agents in MM (the corticosteroid, dexamethasone [DEX] and the IMiD® immunomodulatory drugs, lenalidomide [LEN] and pomalidomide [POM]), as well as in the context of LEN resistance. Single agent CC-223 was shown to inhibit cell proliferation in a panel of 10 MM cell lines achieving IC50 values between 0.1-1 µM following 5 days of treatment. CC-223 also reduced cell viability reaching IC50 values between 0.4-1 µM in 5 out of 10 MM cell lines tested. CC-223 induced concentration-dependent G1 phase arrest within 24h of treatment followed by an induction of cell death by 48h. The anti-MM tumor activity of CC-223 (0.3-10 mg/kg) was further tested in SCID mice with xenotransplants of NCI-H929 grown to approximately 100-150 mm3 in size. A dose-dependent tumor growth inhibition and tumor growth delay was seen with once daily dosing of CC-223. Combination of CC-223 with standard of care therapy compounds was also evaluated in vitro. The combination of CC-223 and DEX demonstrated synergistic effects on the inhibition of cell proliferation in 6 MM cell lines (combination index: 0.0002-0.38) tested over 5 days. CC-223 also had synergistic effects on the same panel of MM cell lines when combined with LEN (combination index: 0.05-0.8). Acquisition of drug resistance in patients receiving standard of care therapies is still one of the major clinical problems in MM. POM, the next generation of IMiD® immunomodulatory agents, has shown clinically meaningful results in patients that are resistant or have relapsed to their drug regimens, including LEN. We have recently developed in vitro cellular models of LEN-resistance using the H929 MM cell line. H929 cells with acquired resistance to LEN (H929 R10-1, R10-2, R10-3 and R10-4) were shown to have one copy number loss of cereblon compared to their matched LEN-sensitive control (H929 D1). In addition to this, protein expression analysis identified that these resistant cell lines also gained the activation of signaling pathways such as PI3K/AKT/mTOR, MEK/MAPK as well as anti-apoptotic factors. For example, S473 AKT phosphorylation was highly elevated in LEN-resistant cell lines which correlated with loss of PTEN protein expression (H929 R10-3 and R10-4). Interestingly, regardless of PI3K/AKT/mTOR pathway status, all LEN-sensitive and resistant H929 cells responded to CC-223 treatment with a strong inhibition of cell proliferation (H929 D1 IC50 0.2 µM, and H929 R10 1-4 IC50 0.2-0.35 µM) and to a lesser effect, induction of cell death, over a 5 day period. Similar to the panel of MM cell lines, G1 arrest occurred after 24h treatment and cell death (Sub-G1) was increased by 72h of treatment. CC-223 treatment reduced S473 pAKT and p-4EBP1 after 1h while total AKT and 4EBP1 remained unchanged in both the sensitive and resistant MM cell lines. Combination treatment of LEN-sensitive and resistant H929 cells with CC-223 and POM had synergistic inhibitory effects on cell proliferation (combination index: 0.35-0.7) and cell viability (combination index: 0.15-0.42). In conclusion, the mTOR kinase inhibitor, CC-223 potently inhibited MM cell proliferation by inducing G1 arrest and cell death in a panel of MM cell lines and reduction of tumor volume in vivo. The combination of LEN, POM or DEX with CC-223 had synergistic effects on MM cell proliferation and viability. Therefore, CC-223 in combination with other standard of care agents could become an important clinical tool for the treatment of MM in the future. Disclosures: Rychak: Celgene Corporation: Employment, Equity Ownership. Mendy:Celgene: Employment, Equity Ownership. Miller:Celgene Corporation: Employment, Equity Ownership. Leisten:Celgene Corporation: Employment, Equity Ownership. Narla:Celgene Corporation: Employment, Equity Ownership. Raymon:Celgene Corporation: Employment, Equity Ownership. Chopra:Celgene: Employment, Equity Ownership. Lopez-Girona:Celgene: Employment, Equity Ownership.

BH3 Profiling As Predictor Of 5-Azacytidine and Decitabine Clinical Responses

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 603-603 ◽  
Author(s):  
Raoul Tibes ◽  
Steven M. Kornblau ◽  
William E. Pierceall ◽  
Ryan Lena ◽  
Yi Hua Qiu ◽  
...  
Keyword(s):  
Cell Lines ◽  
Statistical Significance ◽  
Family Members ◽  
Specific Inhibitor ◽  
Hypomethylating Agents ◽  
Employment Equity ◽  
Number Of Patients ◽  
Clinical Responses ◽  
Equity Ownership

Abstract Hypomethylating agents have changed treatment and outcomes in MDS and are active in AML patients. However a significant number of patients do not respond to 5-Azacytidine or Decitabine and there is no clinically validated assay that predicts response to 5-Azacytidine (5-Aza) or Decitabine (DAC). Clinical parameters and recently TET2 mutations have been identified as genetic predictors of response to 5-Aza. Several recent papers report on the possible predictive value of anti-apoptotic proteins, for example BCL2L10. In previous genome-scale RNA-interference (RNAi) screens anti-apoptotic BCL-2 family members, most potently BCL-XL, were identified as top targets whose down-regulation sensitize to 5-Aza. Results were confirmed with the BCL-XL and BCL-2 inhibitor ABT-737, as well as the BCL-2-specific inhibitor ABT-199, both of which sensitized myeloid cells to 5-Aza. Although it is expected that interfering with anti-apoptotic genes would sensitize to hypomethylating similar to cytotoxics, few of the other >900 genes from our RNAi sensitizer screens, including few of the 571 kinases, sensitized to 5-Aza as potently as inhibition of BCL-XL. Thus BCL-2 family members constitute an especially potent context to modulate the activity of 5-Aza. To assess if protein expression of the top targets BCL-XL, BCL-2 or MCL-1 correlate with and may explain a preferential sensitization, 577 primary AML samples were examined by Reverse Phase Protein Array (RPPA). There was substantial overlap of expression across and within FAB subgroups and cytogenetics, and expression of neither of these genes/proteins could explain the observed effects. This may be expected due to functional redundancies amongst the different anti-apoptotic BCL-2 family members, as well as distinct, yet overlapping binding affinities between anti- and pro-apoptotic BCL-2 proteins and BH3 peptides. Thus, we next aimed to functionally interrogate the overall balance of pro- and anti-apoptotic BCL-2 family members, also described as “primedness” by BH3 profiling. This assay uses peptides derived from pro-apoptotic BH3-only proteins to determine the capacity of cells to initiate apoptosis in response to pro-apoptotic BH3 molecules [anti-apoptotic BCL-2 molecules like BCL-XL and BCL-2 bind and inhibit BH3-only molecules]. This assay is a broad functional readout that incorporates many parameters including the consequences of varying BCL2L10 expression. To establish a proof-of-concept that the functional interrogation of specific apoptotic thresholds may be an optimal readout for 5-Aza response, we first assessed a broad panel of 13 AML-derived cell lines by BH3 profiling and in parallel 5-Aza drug dose response experiments. Several BH3 profiling metrics, including NOXA plus BIM, significantly correlated with 5-Aza sensitivity in vitro (Figure 1). Next, to correlate actual clinical responses to 5-Aza with BH3 profiling, specimens from 28 AML, MDS and MDS/MPN overlap patients treated with 5-Aza or DAC-based regimens and for whom clinical outcome was available, were assayed in BH3 assays. In the clinic, the best BH3 metrics were combined values from NOXA and BIM peptides similar to cell lines. NOXA plus BIM discriminated clinical responses defined as achieving any response (sensitive) versus a patient being resistance/refractory to 5-Aza/DAC based regimens with statistical significance (Mann-Whitney two-tailed p = 0.001). This was true for the entire group of patients (5-Aza- and DAC- regimens) with a Receiver Operating Characteristic (ROC) of an AUC=0.875, with a further increase for patients treated only with 5-Aza based regimens with an AUC=0.95.Figure 1BH3 metrics distinguish cell lines with higher EC 50 (> 2uM) from those with lower EC50 (generally < 1uM).Figure 1. BH3 metrics distinguish cell lines with higher EC 50 (> 2uM) from those with lower EC50 (generally < 1uM). In conclusion, due to significant expression overlap and functional redundancies of the BCL-2 family proteins, expression does not correlate with 5-Aza clinical or in vitro responses. However, BH3 profiling as a general functional readout of apoptotic primedness, significantly discriminated responses in patients as well as in vitro. Thus, BH3 profiling incorporates the entirety of pro- and anti-apoptotic molecules (including BCL2L10) and is promising to predict responses to hypomethylating agents. We propose to prospectively validate BH3 profiling as a predictive biomarker assay for 5-Aza or DAC based regimens. Disclosures: Pierceall: Eutropics: Employment, Equity Ownership. Lena:Eutropics: Employment, Equity Ownership. Cardone:Eutropics: Employment, Equity Ownership. Elashoff:Eutropics: Employment. Blake:Noel Blake: Employment, Equity Ownership.

SL-401, a Novel IL-3Rα/CD123-Directed Agent Targets Stem-like Cells in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4463-4463 ◽  
Author(s):  
Arghya Ray ◽  
Yan Song ◽  
Deepika Sharma Das ◽  
Vincent Macri ◽  
Janice Chen ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Stem Cell ◽  
Board Of Directors ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Drug Resistant ◽  
Employment Equity ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Rpmi 8226

Abstract Introduction Multiple myeloma (MM) remains incurable despite novel therapies, highlighting the need for further identification of factors mediating disease progression and resistance. One such factor is the development of a drug-resistant stem cell-like subpopulation. Previous studies have shown that MM side population cells (MM-SPs) exhibit stem-cell like features and contribute to relapse of MM. Recent research efforts, which have focused the biology of MM stem-like cells in order to derive specific therapy, have shown that stem-cell transcription factor Oct-4 is linked to stemness and chemoresistance. Here we examined the effect of enforced expression of Oct4 in MM cells and MM-SPs on the development of stem cell-like characteristics and drug-resistance in MM. These studies allow us to establish stable MM cell lines with characteristic stem-cell like features, which in turn facilitate screening of novel agents that effectively target this cell population in MM. Methods MM-SPs were isolated from RPMI-8226 cells by flow-cytometry based Hoechst 33342 staining. RPMI-8226 and RPMI-8226-SP cells were transfected with a phOct4-GFP construct (Gerrard et al., Stem Cell 2005, 23:124-133), and selected with G418 (0.5 mg/ml) to derive stable RPMI-8226-Oct4 and RPMI-8226-SP-Oct4 cell lines. Oct-4 expression was confirmed using FACS. Cell viability was analyzed by WST assays. SL-401 is a targeted therapy directed to IL-3Rα/CD123, comprised of recombinant human IL-3 fused to truncated diphtheria toxin. Drug and reagent source: SL-401 was obtained from Stemline Therapeutics; Bortezomib and flow antibodies were purchased from Selleck Chemicals and BD Biosciences, respectively. Statistical significance was derived using GraphPad Prism. Results 1) RPMI-8226 and RPMI-8226-Oct4 cells were analyzed for the expression of surface markers associated with stem cells (CD123/IL-3Rα, CD133 and CD27) by multicolor flow analysis. Oct-4 transfection does not affect the overall CD123 expression in RPMI-8226 cells, as the % MFI-CD123hi in RPMI-8226 versus RPMI-8226-Oct4 remains unchanged. However, the stable selection makes RPMI-8226-Oct4 more clonal in nature (%CD123hi : RPMI8226; 14.9% vs RPMI-8226-Oct4; 60%). 2) A significant increase in the frequency of CD133+ve cells was observed in RPMI-8226-SP-Oct4-tranfected cells versus either RPMI-8226-SP cells or RPMI-8226-Oct-4 cells [RPMI-8226-SP: 6.3%; RPMI-8226-Oct4: 27.8%; RPMI-8226-SP-Oct4: 40%; p< 0.05]. 3) Analysis of CD27 surface marker showed highest expression in RPMI-8226-SP-Oct4 cells compared to RPMI-8226-Oct4, RPMI-8226-SP, or RPMI-8226 cells (% MFI: RPMI-8226-SP-Oct4 > RPMI-8226-Oct4 > RPMI-8226-SP > RPMI-8226 cells). 4) Treatment of RPMI-8226 and RPMI-8226-Oct4 cells with proteasome inhibitor bortezomib decreased the viability of RPMI-8226 cells; in contrast, bortezomib did not significantly alter the viablity of RPMI-8226-Oct4 cells [% Viability after bortezomib: RPMI-8226; <50% versus RPMI-8226-Oct4; 95%]. Finally, 5) SL-401 significantly decreased the viability of RPMI-8226-Oct4 cells [IC50: RPMI-8226-Oct4 cells: 75 pM; RPMI-8226-SP cells: 350 pM; RPMI-8226 cells: 1367 pM]. We have previously shown anti-MM activity of SL-401 by an additional mechanism of targeting IL-3Rα-expressing plasmacytoid dendritic cells (pDCs) localized in the tumor microenvironment and blocking pDC-induced MM cell growth. Conclusions Our data show that stem-like cells in MM are relatively resistant to proteasome inhibitor therapy. Importantly, a novel agent SL-401 effectively targets these cells. Oct4-driven stable RPMI-8226 MM cell line serves as a novel tool to screen and develop newer agents targeting stem-like cells in MM. Overall, we show the ability of SL-401 to target a drug-resistant stem-like cell population in MM, and provide an additional rationale for clinical evaluation of SL-401 to improve patient outcome. A clinical trial of SL-401 in MM is currently ongoing (NCT02661022). Disclosures Macri: Stemline Therapeutics, Inc.: Employment. Chen:Stemline Therapeutics, Inc.: Employment, Equity Ownership. Richardson:Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Brooks:Stemline Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Chauhan:Oncopeptide AB: Consultancy; Epicent Rx: Consultancy; C4 Therapeutics: Equity Ownership; Stemline Therapeutics, Inc.: Consultancy. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Sonofi Aventis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Gilead: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees.

scholarly journals The Combination of Entospletinib and Vincristine Demonstrates Synergistic Activity in a Broad Panel of Hematological Cancer Cell Lines and Anti-Tumor Efficacy in a DLBCL Xenograft Model

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5123-5123 ◽  
Author(s):  
Mark Joseph Axelrod ◽  
Peter Fowles ◽  
Jeff Silverman ◽  
Astrid Clarke ◽  
Jennifer Tang ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Growth Inhibition ◽  
B Cell ◽  
Cell Lines ◽  
Xenograft Model ◽  
Lymphocytic Leukemia ◽  
Employment Equity ◽  
Equity Ownership

Abstract Background Entospletinib (GS-9973) selectively inhibits spleen tyrosine kinase (SYK), a critical signaling component of the BCR pathway that is expressed primarily in cells of hematopoietic lineage including normal and malignant B-lymphocytes. Entospletinib is currently in phase II clinical trials, where it has demonstrated both a high degree of safety as well as efficacy against chronic lymphocytic leukemia (Sharman, J., et al. Blood, 2015) and other B cell malignancies. Despite these successes, new therapeutic options, including combinations with standard of care agents, are needed in order to achieve the goal of curing disease through finite treatment. We show here that the combination of entospletinib and vincristine causes synergistic apoptosis in vitro in a broad panel of cell lines derived from hematological cancers including diffuse large B cell lymphoma (DLBCL), acute lymphocytic leukemia, follicular lymphom), multiple myeloma, and acute myelogenous leukemia. We also evaluated and compared the in vivo efficacy of entospletinib and vincristine as singe agents and in combination in a DLBCL tumor xenograft model using the SU-DHL-10 cell line. Methods In vitro growth inhibition of a panel of malignant hematological cell lines was assessed using CellTiter-Glo™ Assay (Promega) after 72h incubation with entospletinib or vincristine alone or in combination. Synergy was evaluated using the Bliss model of independence (Meletiadis, J., et al., Med Mycol, 2005). In vivo, SU-DHL-10 cells (5 x 106 cells) were implanted subcutaneously in the axilla in male SCID beige mice. All mice were sorted into study groups on Day 16 such that each group's mean tumor volume fell within 10% of the overall mean (197mm3). Dosing was initiated on Day 16 and animals were dosed for 17 days. Plasma concentrations of entospletinib and vincristine were assessed on Day 19, and the entospletinib 75 mg/kg dose was lowered on Day 22 to 50 mg/kg to approximate the human achievable SYK target coverage of EC80. Efficacy and tolerability were evaluated by tumor measurements and body weight monitored three times weekly. Tumor burden data were analyzed by the application of a two-way analysis of variance (ANOVA), with post-hoc analysis. Results In vitro combinations of entospletinib with low concentrations of vincristine resulted in marked inhibition of cell proliferation and induction of apoptosis in a broad panel of 19 tumor cell lines representing major B cell malignancies including DLBCL. The combination of entospletinib with vincristine had a profound inhibitory effect on proliferation in all subtypes of DLBCL. Entospletinib was evaluated at a concentration equivalent to the Cminof the clinical dose and vincristine was used at concentrations (≤ 10 nM) that had little to no significant single agent effect in these cell lines. In vivo in a SU-DHL-10 xenograft model, entospletinib dosed alone at 25 or 75/50 mg/kg significantly inhibited tumor growth, causing 39% and 20% tumor growth inhibition (TGI), respectively, compared to the vehicle-treated control group. Vincristine administered at either 0.15 and 0.5 mg/kg Q7D x 3 also resulted in significant TGI (42% and 85% TGI, respectively). The addition of entospletinib (75/50 mg/kg) to 0.5 mg/kg or 0.15 mg/kg vincristine resulted in a significant increase in TGI from 85% to 96% (p= 0.001) and 42% to 71% (p< 0.0001), respectively. The addition of entospletinib (25 mg/kg) to vincristine did not significantly increase the tumor growth inhibition. While the groups receiving either entospletinib or vincristine as single agents had no complete or partial tumor regression, 50% of the mice receiving the combination of 75/50 mg/kg entospletinib with 0.5 mg/kg vincristine had partial responses, 8% had complete regression and 8% were tumor free at the end of study (Figure 1). Conclusion Entospletinib and vincristine demonstrated efficacy and tolerability both alone and in combination in the SU-DHL-10 DLBCL cell line xenograft model in SCID beige mice. Vincristine combinations with entospletinib showed significantly greater efficacy than vincristine alone. These data support the further clinical development of entospletinib in combination with vincristine for the treatment of DLBCL. a ENTO: PO: Q12H x 2 (Day 16-32) b VCR: IV: Q7D x 3 (Days 18, 25, 32) Figure 1. Tumor Regressions in an Entospletinib/ Vincristine Treated Murine DLBCL Xenograft Figure 1. Tumor Regressions in an Entospletinib/ Vincristine Treated Murine DLBCL Xenograft Disclosures Axelrod: Gilead Sciences: Employment, Equity Ownership. Fowles:Gilead Sciences: Employment, Equity Ownership. Silverman:Gilead Sciences: Employment, Equity Ownership. Clarke:Gilead Sciences: Employment, Equity Ownership. Tang:Gilead Sciences: Employment, Equity Ownership. Rousseau:Gilead Sciences: Employment, Equity Ownership. Webb:Gilead Sciences: Employment, Equity Ownership. Di Paolo:Gilead Sciences: Employment, Equity Ownership.

Dacetuzumab (SGN-40), Lenalidomide, and Weekly Dexamethasone in Relapsed or Refractory Multiple Myeloma: Multiple Responses Observed in a Phase 1b Study.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2870-2870 ◽  
Author(s):  
Edward Agura ◽  
Ruben Niesvizky ◽  
Jeffrey Matous ◽  
Nikhil Munshi ◽  
Mohamad Hussein ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Antitumor Activity ◽  
Dose Escalation ◽  
Research Funding ◽  
Low Dose ◽  
Employment Equity ◽  
Phase 1B ◽  
Equity Ownership ◽  
Expansion Cohort ◽  
Number Of Cycles

Abstract Abstract 2870 Poster Board II-846 Background: Dacetuzumab (SGN-40) is a humanized monoclonal antibody that targets CD40. In prior phase 1 studies, antitumor activity was demonstrated with dacetuzumab monotherapy in patients with non-Hodgkin lymphoma and stabilization of disease was noted in multiple myeloma (MM) patients. Preclinical data demonstrate that the addition of lenalidomide to dacetuzumab markedly enhances dacetuzumab-mediated ADCC and apoptosis in both MM cell lines and patient isolates through modulation of NK cell activity. Methods: A phase 1b, dose-escalation study of dacetuzumab, lenalidomide, and low-dose dexamethasone has been initiated in patients with relapsed or refractory MM. Patients were enrolled in a 3+3 dose-escalation scheme and were given cohort-specific doses of weekly IV dacetuzumab (4, 8, or 12 mg/kg) in addition to oral lenalidomide (25 mg, Days 1-21 every 28 days), and weekly oral dexamethasone (40 mg). At the conclusion of dose escalation, the highest tolerated dose combination was used to treat an expansion cohort, approximately half of whom were lenalidomide-naïve. The maximum number of cycles permitted was 8, or 2 cycles beyond a complete response (CR). Results: Thirty-six patients from 8 centers were enrolled and treated, 15 patients in 3 dose-escalation cohorts and 21 patients in the expansion cohort at the 12 mg/kg dacetuzumab dose level. The median age of patients was 65 years (range 48-83) with a median of 3.4 years since diagnosis (range 1-11); patients were heavily pretreated (median of 4 prior systemic regimens [range 2-14]). The median number of cycles received was 4 (range 1-8), although 14 patients remain on treatment. The most common adverse events (AEs) in the study were fatigue (47%), neutropenia (28%), thrombocytopenia (25%), diarrhea (22%), constipation (19%), and headache (19%), the majority of which were Grade 1 or 2 in severity. Two patients discontinued treatment due to dose-limiting toxicity (DLT) during Cycle 1: one patient developed Grade 3 herpes zoster (4 mg/kg); a second patient developed Grade 4 renal failure requiring dialysis (12 mg/kg). This patient had IgA myeloma with a rising M-protein and deteriorating kidney function at study entry; the patient's renal function eventually improved and dialysis was no longer required. Another patient discontinued treatment due to a serious AE that was not considered a DLT (tumor lysis syndrome). Of 33 patients who have had Investigator-assessed response evaluations according to the European Group for Bone Marrow and Transplantation criteria, 13 patients (39%) achieved an objective response (1 CR, 12 partial responses [PR]); other responses were 4 (12%) minimal responses (MR), 10 patients (30%) had stable disease (SD), 2 patients (6%) had progressive disease (PD), and 4 patients (12%) were not evaluable. Responses that occurred among lenalidomide-naïve patients (n=18) were 1 CR, 9 PR, 1 MR, 3 SD, 2 PD, and 2 not evaluable. Among patients who have previously received lenalidomide (n=18), the responses were 3 PR, 3 MR, 7 SD, and 2 not evaluable; additionally, 3 patients who are receiving treatment have not yet been assessed for response. Conclusion: The combination regimen of dacetuzumab, lenalidomide, and low-dose dexamethasone was generally well tolerated at all doses studied. Evidence of anti-myeloma activity with this combination is encouraging as antitumor activity was observed in patients with MM who have previously received lenalidomide as well as lenalidomide-naïve patients. Disclosures: Agura: Seattle Genetics, Inc.: Research Funding. Off Label Use: Dacetuzumab is an investigational agent. Niesvizky:Proteolix: Research Funding, data monitoring committee; Seattle Genetics, Inc: Research Funding; Millenium: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau. Matous:Seattle Genetics, Inc.: Research Funding; Celgene: Honoraria, Speakers Bureau. Munshi:Celgene: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics, Inc.: Research Funding. Hussein:Seattle Genetics, Inc.: Research Funding. Parameswaran:Seattle Genetics, Inc.: Research Funding. Tarantolo:Seattle Genetics, Inc.: Research Funding. Whiting:Seattle Genetics, Inc.: Employment, Equity Ownership. Drachman:Seattle Genetics, Inc.: Employment, Equity Ownership. Zonder:Pfizer: Consultancy; Seattle Genetics, Inc.: Research Funding; Millennium: Research Funding; Amgen: Consultancy; Celgene: Speakers Bureau.

scholarly journals SL-801, a Novel, Reversible Inhibitor of Exportin-1 (XPO1) / Chromosome Region Maintenance-1 (CRM1) with Broad and Potent Anti-Cancer Activity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4433-4433
Author(s):  
Janice Chen ◽  
Christopher L. Brooks ◽  
Peter McDonald ◽  
Jonathan D. Schwartz ◽  
Rebecca S. Schneider ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Cell Lines ◽  
Nuclear Export ◽  
Xenograft Model ◽  
Therapeutic Index ◽  
Tumor Cell Lines ◽  
Employment Equity ◽  
Anti Cancer ◽  
Xenograft Models ◽  
Equity Ownership

Abstract XPO1/CRM1, the principal nuclear export protein in eukaryotic cells, is required for the nuclear-cytoplasmic transport of both proteins and RNAs. Overexpression of XPO1 is reported in many cancers, causing dysregulated protein localization, aberrant cell proliferation, and resistance to apoptosis, and is associated with aggressive characteristics and poor patient outcome. Recent work has revealed XPO1 to be a clinically relevant target, and nuclear export inhibitors have emerged as a new class of anti-cancer agents with clinical activity in multiple hematologic and solid malignancies. SL-801 is a novel small molecule that binds covalently to Cys528 of XPO1, blocking the ability of XPO1 to interact with substrate cargos (e.g., p53, FOXO, p21, p27, and others). In contrast to the prototypical XPO1 inhibitor leptomycin B, which binds irreversibly to XPO1 and caused significant toxicities in Phase 1 trials, SL-801 binding to XPO1 is reversible, a characteristic that may be exploited to maximize its therapeutic index. Exposure to SL-801 results in potent inhibition of XPO1-dependent nuclear export, cell cycle arrest, and induction of apoptosis in a time- and dose-dependent manner. Here, the anti-tumor activity of SL-801 was investigated against a panel of 240 cell lines representing a broad range of solid and hematologic malignancies and confirmed in several SCID xenograft models. The OncoPanelTM high content screening platform was used to evaluate the cytotoxicity of SL-801 against 205 solid tumor and 35 liquid tumor cell lines. SL-801 demonstrated potent activity, with 50% growth inhibitory (GI50) values ≤ 10 nM in 51/240 (21.3%) cell lines and GI50 values ≤ 100 nM in 230/240 (95.8%) cell lines. SL-801 sensitivity was independent of cell proliferation rate or XPO1 expression levels. While SL-801 was broadly cytotoxic, cell lines of hematopoietic origin exhibited greater sensitivity. GI50s in hematologic cancers ranged from 3-93 nM in leukemias, 1-103 nM in lymphomas, and 3-11 nM in multiple myelomas. SL-801 also inhibited solid tumor growth, with GI50s ≤ 10 nM in several breast, brain, cervical, ovarian, gastric, kidney, liver, lung, melanoma, prostate, and sarcoma lines. In addition, a 5-fold increase in active caspase-3 staining was observed at SL-801 concentrations ≤ 100 nM in 117/240 (48.8%) cell lines, consistent with induction of apoptosis. To understand tumor sensitivity to SL-801, results of the cell line cytotoxicity screen were analyzed against publicly available genomic datasets. This analysis revealed that SL-801 was cytotoxic towards cell lines regardless of mutation status of key oncogenes (e.g., KRAS) and tumor suppressor genes (e.g., TP53). The in vitro cytotoxicity of SL-801 against tumor cell lines was further validated in several xenograft models in SCID mice. In the RPMI-8226 multiple myeloma xenograft model, tumor growth was significantly inhibited at oral SL-801 doses of 31.25 mg/kg administered daily for five days for two weeks. In the ARH-77 human multiple myeloma xenograft model, overall survival was significantly prolonged by daily oral administration of 125 mg/kg SL-801 for ten days. This dose and regimen were well tolerated, and 90% of SL-801-treated mice survived > 150 days, whereas median survival was 39.5 days in the vehicle-treated group (p < 0.001). Significant tumor growth inhibition was also observed in the NCI-H226 non-small cell lung cancer and 22RV1 prostate cancer xenograft models. These data demonstrate that SL-801 is a promising clinical candidate that inhibits a novel, clinically validated target and support its clinical development in a broad range of oncologic indications. The reversible binding of SL-801 to XPO1 may offer the potential to develop dosing schedules to enable recovery in normal tissues, thus broadening the therapeutic index of this class of agents. IND-enabling work is underway to support entry into clinical trials, and a Phase I trial design will be discussed. Disclosures Chen: Stemline Therapeutics, Inc.: Employment, Equity Ownership. Brooks:Stemline Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. McDonald:Stemline Therapeutics, Inc.: Employment, Equity Ownership. Schwartz:Stemline Therapeutics, Inc.: Employment, Equity Ownership. Schneider:Stemline Therapeutics, Inc.: Employment. Sakakibara:CanBas Co., Ltd.: Employment, Equity Ownership. Saito:CanBas Co., Ltd.: Employment, Equity Ownership. Sato:CanBas Co., Ltd.: Employment, Equity Ownership, Patents & Royalties. Kawabe:CanBas Co., Ltd.: Employment, Equity Ownership, Patents & Royalties. Rowinsky:Stemline Therapeutics: Employment, Equity Ownership.

scholarly journals 5-Azacytidine Induces NOXA and PUMA Expression to Prime AML Cells for Venetoclax-Mediated Apoptosis

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2644-2644
Author(s):  
Dan Cojocari ◽  
Sha Jin ◽  
Julie J Purkal ◽  
Relja Popovic ◽  
Nari N Talaty ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Cell Lines ◽  
Acute Treatment ◽  
Low Dose ◽  
Dna Demethylation ◽  
Phase Iii ◽  
Employment Equity ◽  
Phase Iii Clinical Trial ◽  
Equity Ownership

Abstract Acute myeloid leukemia (AML) is a clonal hematologic malignancy characterized by genomic heterogeneity and epigenetic changes, including aberrant DNA hypermethylation. Phase-Ib clinical data in relapsed/refractory AML patients indicate that combining venetoclax with the hypomethylating agents (HMAs) 5-azacitidine (5-Aza) or decitabine results in an overall response (OR) of 62% (DiNardo et al. 2018) compared to the historical OR of 28-29% with HMAs treatment alone (Kantarjian et al. 2013; Dombret et al. 2015). Subsequently, a randomized phase-III clinical trial was initiated to evaluate venetoclax activity in combination with 5-Aza in treatment-naïve AML patients ineligible for standard induction therapy (M15-656, NCT02993523). However, the underlying mechanism for the combinational activity observed between venetoclax and 5-Aza is unknown. In this study, we demonstrate that both chronic low-dose 5-Aza treatment, which induced global DNA demethylation, and acute treatment (24 hours, non-epigenetic effects) can drive combinational activity with venetoclax in AML through distinct mechanisms. Chronic culture with a low-dose 5-Aza for one week sensitized AML cell lines to venetoclax in vitro. In contrast, acute treatment with 5-Aza, activated the integrated stress response (ISR) pathway to induce expression of the BH3-only proteins NOXA (PMAIP1) and PUMA (BBC3) in human AML cell lines, independent of DNA methylation. This resulted in an increase in the amount of NOXA and/or PUMA in complex with anti-apoptotic proteins like BCL-2, BCL-XL and MCL-1, thereby "priming" AML cells for induction of apoptosis by venetoclax treatment. Priming for apoptosis resulted in significant synergistic cell death in a panel of AML cell lines treated with venetoclax and 5-Aza in vitro. In this panel of cell lines, the level of the PMAIP1, BBC3, and DDIT3 gene induction correlated with the synergy observed between venetoclax and 5-Aza. Importantly, subsequent PMAIP1 deletion significantly impacted the kinetics and depth of apoptosis induced by 5-Aza or venetoclax alone or in combination. In accordance with the in vitro combinational activity, the venetoclax/5-Aza combination provided added benefit over either agent alone in two xenograft models of AML. Together, these data provide a rationale for an ongoing randomized phase-III clinical trial evaluating venetoclax activity in combination with 5-Aza (M15-656, NCT02993523). Disclosures: DC, SJ, JP, RP, NT, YX, EB, JL, and DP are employees of AbbVie. LS is a former employee of AbbVie and was employed during the duration of this study. The design, study conduct, and financial support for this research were provided by AbbVie and Genentech. AbbVie participated in the interpretation of data, review, and approval of the publication. Disclosures Cojocari: AbbVie Inc: Employment. Jin:AbbVie Inc: Employment, Equity Ownership. Purkal:AbbVie Inc: Employment, Equity Ownership. Popovic:AbbVie Inc: Employment, Equity Ownership. Talaty:AbbVie Inc: Employment, Equity Ownership. Xiao:AbbVie Inc: Employment, Equity Ownership. Solomon:AbbVie Inc: Equity Ownership. Boghaert:AbbVie Inc: Employment, Equity Ownership. Leverson:AbbVie Inc: Employment, Equity Ownership, Patents & Royalties. Phillips:AbbVie Inc: Employment, Equity Ownership, Patents & Royalties.

SGN-CD33A: A Novel CD33-Directed Antibody-Drug Conjugate, Utilizing Pyrrolobenzodiazepine Dimers, Demonstrates Preclinical Antitumor Activity Against Multi-Drug Resistant Human AML

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3589-3589 ◽  
Author(s):  
May S.K. Sutherland ◽  
Roland B. Walter ◽  
Scott C. Jeffrey ◽  
Patrick J. Burke ◽  
Changpu Yu ◽  
...  
Keyword(s):  
Single Dose ◽  
Multidrug Resistance ◽  
Antitumor Activity ◽  
Tumor Growth ◽  
Cell Lines ◽  
Tumor Regression ◽  
Drug Resistant ◽  
Antibody Drug Conjugate ◽  
Drug Conjugate ◽  
Antibody Drug

Abstract Abstract 3589 Outcomes for patients with acute myeloid leukemia (AML) are poor, highlighting the need for novel treatment options. Most AML cells express the myeloid differentiation antigen CD33, making CD33-targeted therapy a potential treatment strategy. Gemtuzumab ozogamicin (GO), an anti-CD33 monoclonal antibody (mAb) conjugated to the cytotoxic agent calicheamicin, has recently been shown to improve survival in newly diagnosed patients with more favorable-risk AML but has insufficient activity in those with poor prognostic features as well as in relapsed disease. Here we report the preclinical testing of a novel CD33-directed antibody-drug conjugate, SGN-CD33A, consisting of a humanized anti-CD33 mAb with 2 engineered cysteine residues through which pyrrolobenzodiazepine (PBD) dimer drug moieties are conjugated via a maleimidocaproyl valine-alanine dipeptide linker. PBD dimers exert their biological activity by covalent binding and interstrand cross-linking of DNA. Fluorescence microscopy studies showed that SGN-CD33A is rapidly internalized and traffics to lysosomes within hours of binding to CD33-positive AML cell lines. Following uptake, SGN-CD33A induces DNA damage as measured by phosphorylation of histone 2AX, subsequently leading to G2-M cell cycle arrest, disruption of mitochondrial membrane integrity, increased caspase-3 activity, formation of cleaved poly ADP-ribose polymerase, DNA fragmentation and cell death. The anti-leukemic activity of SGN-CD33A was assessed in cytotoxicity assays against 12 AML cell lines and 18 primary AML patient samples of mixed cytogenetic origin (favorable, intermediate and unfavorable) and multidrug resistance (MDR) status. SGN-CD33A was highly active against all AML cell lines tested (mean IC50, 22 ng/ml), including 5 of 5 MDR-positive cell lines (mean IC50, 27 ng/mL). In contrast, GO was moderately active in 1 of 5 MDR-positive cell lines (IC50, 227 ng/mL) but inactive against the other 4 (IC50, >1000 ng/mL). SGN-CD33A was also active against 15 of 18 primary samples isolated from untreated AML patients at diagnosis (mean IC50 of responsive samples, 8 ng/mL) and was more potent than GO which was active in 10 of 18 AML samples (mean IC50 of responding samples, 27 ng/mL). The 3 AML specimens that were resistant to SGN-CD33A each had low or absent CD33 expression, as determined by flow cytometry. Cytogenetic abnormalities and MDR activity did not correlate with in vitro SGN-CD33A cytotoxicity. In vivo antitumor activity was evaluated in AML mouse xenograft models established with MDR-negative HL-60 and MDR-positive THP-1 and TF1-α cell lines. SGN-CD33A dosed once at 300 mcg/kg yielded durable complete regressions in THP-1 (Figure A) and TF1-α xenografts. In these models of drug-resistant AML, treatment with a single dose of 100 mcg/kg SGN-CD33A significantly delayed tumor growth compared to untreated and non-binding control ADC-treated mice (p<0.001), whereas GO was inactive even when dosed at 1000 mcg/kg. In the MDR-negative HL-60 model, a single dose of 30 mcg/kg of SGN-CD33A delayed tumor growth and 100 mcg/kg induced complete tumor regression (Figure B). In contrast, GO had minimal activity when dosed at 100 mcg/kg but did result in durable tumor regression when administered at a ten-fold higher dose (Figure B). Together, these data demonstrate that SGN-CD33A exhibits antitumor activity against a broad panel of primary AML samples and results in durable remissions in preclinical models of MDR-positive AML that are characteristically resistant to conventional chemotherapy and GO. CD33-directed delivery of PBD dimers may overcome multidrug resistance and may represent a new strategy for the treatment of patients with AML. Clinical trials are planned to further evaluate SGN-CD33A in AML. Disclosures: Sutherland: Seattle Genetics, Inc.: Employment. Walter:Seattle Genetics, Inc.: Consultancy, Research Funding. Jeffrey:Seattle Genetics, Inc.: Employment. Burke:Seattle Genetics, Inc.: Employment. Yu:Seattle Genetics, Inc.: Employment. Stone:Seattle Genetics, Inc.: Employment. Ryan:Seattle Genetics, Inc.: Employment. Sussman:Seattle Genetics, Inc.: Employment. Zeng:Seattle Genetics, Inc.: Employment. Benjamin:Seattle Genetics, Inc.: Employment. Bernstein:Seattle Genetics, Inc.: Consultancy. Senter:Seattle Genetics, Inc.: Employment. Drachman:Seattle Genetics, Inc.: Employment. McEarchern:Seattle Genetics, Inc.: Employment.

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