ABT-806 derived antibody drug conjugates (ADCs) inhibit growth of malignant mesothelioma in-vivo.

e14677 Background: Malignant mesothelioma (MM) is an aggressive malignancy of the pleura with limited therapeutic options, and is associated with a poor prognosis. EGFR is known to be highly over-expressed in mesothelioma with reported EGFR overexpression between 44 to 97%. We have investigated an anti-EGFR antibody (ABT-806), which is tumor specific and robustly inhibits EGFR-expressing tumors. We have previously shown that ABT-806 ADCs demonstrate potent anti-tumor activity in 806 immunohistochemistry (IHC) positive MSTO-211H MM cell line xenograft model. We present data in MM using ABT-806 novel ADCs [ABT-414 (ABT-806- monomethyl auristatin F), ABBV-221 (ABT-806- monomethyl auristatin E) and ABBV-321 (ABT-806- pyrrolobenzodiazepine)] in MM patient derived xenografts (PDX). Methods: We evaluated expression of EGFR and mAb 806 IHC in MM cell lines and PDXs. PDXs were implanted into 5 to 10 NOD-Scid mice per group and treated with control ADC, saline, cisplatin or ABT-806 ADCs and followed longitudinally with caliper measurements. Comparative statistics were performed in Graphpad prism. Results: Three PDX models were selected according to their 806 IHC statuses (2 epithelioid 806 IHC positive, 1 biphasic histology 806 IHC negative). In one epithelioid PDX model, ABBV-321 resulted in significantly reduced tumor growth on day 27 post therapy with median tumor volumes of 180 mm3 (ADC control) compared with 78mm3 (ABBV-321; p = 0.0159 two-sided). Moreover, the median survival was also significantly longer in ABBV-321 treated models (p = 0.018). In the other epithelioid PDX model, ABBV-321 also resulted in significant responses (median 428mm3 (ADC control) vs 167mm3 (ABBV-321, p = 0.0201). In the 806 IHC negative PDX model, the differences in tumor volumes between all groups were found to be non-statistically significant (ADC control vs ABT-414, ADC control vs ABBV-221, ADC-control vs ABBV-321 groups) with p = 0.0597 for one-way ANOVA. MSTO211H cell line xenograft model also demonstrated significant anti-tumor response to both ABT-414 and ABBV-221 (p < 0.01). Conclusions: In a disease with limited therapies, ABT-806 targeting ADCs in MM demonstrated significant responses in 806+ PDX and cell lines. These data support clinical expansion of these compounds in 806+ MM patients.

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A Humanized Anti-Ganglioside GM2 Antibody, BIW-8962, Exhibits ADCC/CDC Activity against Multiple Myeloma Cells and Potent Anti- Tumor Activity in Mouse Xenograft Models.

Blood ◽

10.1182/blood.v112.11.1718.1718 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1718-1718 ◽

Cited By ~ 2

Author(s):

Toshihiko Ishii ◽

Asher Alban Chanan-Khan ◽

Jazur Jafferjee ◽

Noreen Ersing ◽

Takeshi Takahashi ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Phase 1 ◽

Xenograft Model ◽

Surface Expression ◽

Scid Mice ◽

Subcutaneous Xenograft ◽

Anti Tumor Activity

Abstract BIW-8962 is a humanized anti-ganglioside GM2 (GM2) monoclonal antibody, produced by Poteligent technology to enhance ADCC activity. GM2 is expressed on many cancer cells including multiple myeloma (MM), small cell lung cancer and glioma cells. In this study, we evaluated the anti-myeloma activity of BIW-8962 in preclinical myeloma models both in vitro and in vivo. Expression of GM2 was analyzed in 15 human MM cell lines by FCM. Eleven out of 15 MM cell lines had positive surface expression of GM2. GM2 as a potential target was then verified in primary MM samples obtained from patients. Eleven out of 15 samples were positive for GM2. We then used two GM2 positive MM cell lines (U266B1 and KMS-11) and evaluated ADCC and CDC activity of BIW-8962 in vitro. BIW-8962 exhibited a potent ADCC and less potent CDC activity. In vivo anti-tumor activity of BIW-8962 was then examined using the standard subcutaneous xenograft model; KMS-11 was inoculated in the flank of SCID mice. BIW-8962 (intravenously administered biweekly for 3 weeks) exhibited a potent anti-tumor activity from as low a dose level as 0.1 mg/kg. Furthermore, in a more clinically relevant model, in which OPM-2/GFP (GM2 positive MM cell line) cells were intravenously inoculated into SCID mice with preferentially tumor growth within the bone marrow microenvironment, BIW-8962 (intravenously administered biweekly for 4 weeks, 10 mg/kg) suppressed OPM-2/GFP cell growth and serum M protein elevation, demonstrating in vivo anti-myeloma effect of BIW-8962. Our preclinical investigations rationalize clinical evaluation of BIW-8962 in patients with MM. Currently BIW-8962 is being investigated in a Phase 1 study in patients with multiple myeloma.

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Evaluation of Two Novel Therapeutics Against Human and Canine Osteosarcoma

10.21203/rs.3.rs-51104/v1 ◽

2020 ◽

Author(s):

Heather Wilson-Robles ◽

Tasha Miller ◽

Chao Sima ◽

Jianping Hua ◽

Milana Cypert ◽

...

Keyword(s):

Cell Line ◽

Cell Lines ◽

Xenograft Model ◽

Western Blots ◽

Primary Bone Tumor ◽

Murine Xenograft Model ◽

Primary Bone ◽

Number Of Cells

Abstract Background: Osteosarcoma (OS) is the most common primary bone tumor in both humans and canines. This tumor has an aggressive course leading to the development of metastatic lesions in most patients diagnosed with this disease. Two new novel agents, MLN9708 and SH4-54, work as a proteasome inhibitor and a STAT3 inhibitor, respectively. Targets of these drugs have been shown to be overexpressed in OS in both species. Methods: Two human and two canine OS cell lines were exposed in vitro to both drugs alone and in combination. The number of cells undergoing apoptosis, as well as the number of cells capable of invasion through a matrigel basement membrane was evaluated after exposure to the drugs. Additionally, PCR and Western blots of downstream targets were evaluated. Finally, both drugs were tested against each cell line in an in vivo murine xenograft model. Results: All four cell lines were sensitive to MLN9708, one of the human cell lines and both canine cell lines were resistant to SH4-54. MLN9708 was also better at inhibiting invasion in three of the four cell lines. In the murine xenografts MLN9708 inhibited growth and metastasis in 143B (human OS) and the combination inhibited growth in the canine OS cell line (MCKOS). Conclusions: Though SH4-54 demonstrated robust cell killing in 143B in vitro, MLN9708 demonstrated broader activity across species for the treatment of OS. Further investigation into this drug is warranted as a treatment for OS. Combination of this drug with a STAT3 inhibitor may be worthwhile in canine OS.

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Characterization of the Effects of Pim Kinase Inhibition on Multiple Oncogene-Driven Cell Lines

Blood ◽

10.1182/blood.v112.11.2662.2662 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2662-2662

Author(s):

Stefan David Gross ◽

John E Robinson ◽

Shelley Allen ◽

April Cox ◽

Walt E DeWolf ◽

...

Keyword(s):

Tumor Growth ◽

Cell Line ◽

Protein Kinases ◽

Cell Lines ◽

Biological Effects ◽

Xenograft Model ◽

Dependent Manner ◽

Kinase Inhibition ◽

In Cells

Abstract We report here the biological effects of inhibiting Pim-1, -2 and -3 serine/threonine protein kinases in cells and in vivo using a novel, potent and selective small molecule inhibitor directed against these kinases. In vitro enzyme assays revealed potent inhibition of all three Pim kinase isoforms while maintaining selectivity against more than 220 other protein kinases. In cells, compound treatment produced biological effects consistent with Pim inhibition, including a reduction in BAD protein phosphorylation. Moreover, this biological activity corresponds well with the compound’s anti-proliferative and pro-apoptotic activity in the IL-3 dependent mouse pro-B cell line Ba/F3 and in factor-indpendent Ba/F3 cell lines dependent upon the expression of BCR-Abl or TEL-JAK2 for growth and survival. Importantly, compared to the BCR-Abl driven Ba/F3 cell line, this compound was equally effective at blocking the growth of an Imatinib-resistant BCR-Abl[ T315I]-driven Ba/F3 cell line. Finally, this compound possesses pharmacokinetic properties that predict the potential for in vivo activity. Therefore, we assessed the effect of Pim kinase inhibition on tumor growth in a mouse subcutaneous tumor xenograft model employing a cell line driven by the TEL-JAK2 oncogene. Results demonstrated that the compound significantly inhibited tumor growth in a dose-dependent manner. In terms of potential clinical utility, these data show that Pim kinase inhibition could be an effective therapeutic strategy for numerous hematologic malignancies including chronic and acute myelogenous leukemias, particularly in those patients that have become resistant to existing therapies such as Imatinib.

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Engineering High Affinity and Cleavage Resistant CD16 to Augment ADCC of Placental Hematopoietic Stem Cells-Derived Natural Killer Cells

Blood ◽

10.1182/blood-2019-127009 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1576-1576

Author(s):

Xuan Guo ◽

Srinivas Somanchi ◽

Rohit Mathur ◽

Shuyang He ◽

Qian Ye ◽

...

Keyword(s):

Nk Cells ◽

Cell Lines ◽

Xenograft Model ◽

Lymphoma Cell ◽

Hematopoietic Stem ◽

High Affinity ◽

Igg Binding ◽

Anti Tumor Activity

Background: Celularity, Inc. is developing human placental hematopoietic stem cells-derived, cryopreserved, off-the-shelf, ex-vivo expanded and allogenic natural killer (PNK) cells for various hematological malignancies and solid tumors. NK cells play a central role in antibody dependent cell mediated cytotoxicity (ADCC) through Fc receptor CD16 in monoclonal antibody mediated anti-tumor therapies. Two allelic forms of CD16 have been identified. The 158Val/Val form has shown to have higher IgG binding affinity compared to the 158Phe/Phe form.1 The high IgG binding allele are found in about 10-20% of the normal population.2,3 In addition, activation of NK cells induces CD16 shedding by matrix metalloprotease ADAM17 at 197Ser, thus limiting ADCC responses. A single mutation (Ser197Pro) prevents CD16 shedding and increases ADCC activity in NK cells.4 Since the antibody binding affinity and CD16 expression of PNK could vary with different donors, we hypothesize that expressing a high affinity (158Val) and proteinase cleavage resistant (197Pro) CD16 variant (CD16VP) augments anti-tumor ADCC activity. Methods: Lentivirus expressing CD16VP was used to transduce human placental CD34+ cells. After transduction, the cells were cultured in the presence of cytokines including thrombopoietin, SCF, Flt3 ligand, IL-7, IL-15 and IL-2, for 35 days to generate PNK-CD16VP cells. Non-transduced PNK cells (NT) served as a control. Expression of CD16VP was evaluated by activating cells with PMA/ionomycin to induce CD16 cleavage (CD16 shedding assay) followed by immunostaining with CD16 antibody and analyzed using flowcytometry. ADCC of PNK-CD16VP cells was assessed against Daratumumab (anti-CD38) or Rituximab (anti-CD20) opsonized lymphoma cell lines at various effector to target (E:T) ratios. IgG was used as ADCC control. In vivo anti-tumor activity was assessed in a Daudi disseminated Xenograft model in NSG mice. Luciferase-expressing Daudi cells (3x106) were intravenously (IV) administered at day 0, followed by PNK-CD16VP cells (10x106) IV at day 1 and day 3, and Daratumumab at day 3. Tumor burden in mice was monitored by Bioluminescence Imaging (BLI). Statistical differences between the groups were calculated using paired t-test using Prism. Results: Lentiviral transduction of CD16VP achieved high expression efficiency in multiple placental CD34+ donors. These cells expanded [7095 ± 2998 folds (n=8)] and differentiated into PNK cells (>90% CD56+CD3-) at day 35. PNK-CD16VP expressed 64.6 ± 10.3% (n=8) of CD16, while the NT expressed 12.1 ± 3.3% (n=8) CD16. PMA/ionomycin induced >89% shedding of CD16 in NT cells, while significantly less (<11%) CD16 shedding was observed in PNK-CD16VP cells. These results indicated that CD16VP was expressed and maintained throughout the culture process. In vitro ADCC assay demonstrated improved anti-tumor activity of PNK-CD16VP cells over NT cells against Daratumumab or Rituximab opsonized lymphoma cell lines. At 10:1 E:T ratio PNK-CD16VP cells elicited higher cytotoxicity compared to NT: 47 ± 13% against Daratumumab opsonized Daudi cells versus 25 ± 5% (n=5; p<0.05); 30 ± 13% against Daratumumab opsonized HS-Sultan cells versus 21 ± 14% (n=3; p<0.05); 30 ± 7% against Daratumumab opsonized Sudhl6 cells versus 16 ± 10% (n=3; p<0.05). Improved ADCC activities in PNK-CD16VP were also observed in other cell lines including Raji and Sudhl4 with Daratumumab and Rituximab antibodies. PNK-CD16VP were used to test anti-tumor ADCC in vivo using a disseminated Daudi Xenograft model. The preliminary data demonstrated that PNK-CD16VP combined with Daratumumab reduced BLI signal (>50%) compared to vehicle or Daratumumab alone at day 10 after treatment. This observation suggested that PNK-CD16VP demonstrated in vivo ADCC anti-tumor activity. Conclusions: In this study, we genetically modified PNK to express high affinity and cleavage resistant CD16 variant using lentivirus. The PNK-CD16VP cells demonstrated enhanced ADCC function against lymphoma cell lines in vitro and in vivo. Further development of PNK-CD16VP for immune-oncology therapeutics is warranted. References: Wu J et al. J Clin Invest. 1997;100(5):1059-1070.Sugita N et al. Clin Exp Immunol. 1999;117(2):350-354.Koene HR et al. Blood. 1997;90(3):1109-1114.Jing Y et al. PLoS One. 2015;10(3):e0121788. Disclosures Guo: Celularity, Inc.: Employment. Somanchi:Celularity Inc: Employment. Mathur:Celularity Inc: Employment. He:Celularity Inc: Employment. Ye:Celularity Inc: Employment. Difiglia:Celularity Inc: Employment. Rotondo:Celularity Inc: Employment. Rana:Celularity Inc: Employment. Ling:Celularity Inc: Employment. Edinger:Celularity Inc: Employment. Hariri:Celularity Inc: Employment. Zhang:Celularity Inc: Employment.

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Anti-tumor activity of ADH-1 in vitro and in vivo in combination with paclitaxel in ovarian cancer cell lines

Journal of Clinical Oncology ◽

10.1200/jco.2007.25.18_suppl.16050 ◽

2007 ◽

Vol 25 (18_suppl) ◽

pp. 16050-16050

Author(s):

M. Gupta ◽

D. Barnes ◽

J. Losos ◽

G. Spehar ◽

M. Bednarcik ◽

...

Keyword(s):

Ovarian Cancer ◽

Combination Therapy ◽

Tumor Cells ◽

Cell Lines ◽

Cancer Cell ◽

Xenograft Model ◽

In Vitro Cytotoxicity ◽

Anti Tumor Activity

16050 Background: ADH-1 is a novel N-cadherin (Ncad) antagonist. Ncad is a protein present on certain tumor cells and established tumor blood vessels. Its expression on tumor cells increases as they become more aggressive, invasive and metastatic, making it an important target for anti-cancer therapy. ADH-1 was well tolerated in phase I studies and demonstrated evidence of anti-tumor activity in 7 patients whose tumors expressed Ncad. Patient enrollment in two phase II single agent trials concluded at the end of 2006. We report on the anti-tumor activity of ADH-1 in combination with paclitaxel in cancer cell lines in vitro and in the A2780 (Ncad positive) ovarian xenograft model in vivo. Methods: In vitro cytotoxicity of SKOV-3 (ovarian) cells exposed to a fixed ratio of ADH-1 and paclitaxel simultaneously was evaluated by the WST-1 cell proliferation assay. In vivo anti-tumor activity of ADH-1, paclitaxel, and the combination was evaluated in the A2780 xenograft model. ADH-1 100 mg/kg was administered bid IP for 21 days and paclitaxel was administered qod IV for 5 days. Results: In vitro cytotoxicity assays evaluated for combination effects using CalcuSyn software indicated a strong synergistic effect of ADH-1 in combination with paclitaxel (CI <1). In vivo paclitaxel treatment produced a median Time to Endpoint (TTE) (tumor volume >2gm or study end at 60 day) of 32.1 days and 73% Tumor Growth Delay (TGD), compared to control (p=0.028). For the paclitaxel group, there was only one Tumor Free Survivor (TFS) and one transient Complete Responder (CR). ADH-1 produced a TTE of 16.1 and a -13% TGD (p>0.05). The combination of ADH-1 and paclitaxel produced a median TTE of 48.6 days, corresponding to 161% TGD (p<0.0016 compared to untreated controls, p<0.003 for vehicle treated, and p<0.005 compared to paclitaxel alone). The combination therapy generated durable CR in 5 animals, 1 transient CR and 2 PR. The combination therapy had similar toxicity to paclitaxel alone. Conclusions: In this ovarian cancer model, the combination of ADH-1 with paclitaxel produced a synergistic anti-tumor effect. Based in part on these encouraging pre-clinical results, a clinical program of ADH-1 in combination with chemotherapeutic agents has been initiated. No significant financial relationships to disclose.

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OTME-14. TGF-beta signaling in microtube formation of glioblastoma

Neuro-Oncology Advances ◽

10.1093/noajnl/vdab070.065 ◽

2021 ◽

Vol 3 (Supplement_2) ◽

pp. ii16-ii16

Author(s):

Simon Storevik ◽

Justin Joseph ◽

Capucine Magaut ◽

Luiz Henrique ◽

Thomas Mathivet ◽

...

Keyword(s):

Cell Line ◽

Rna Sequencing ◽

Cell Lines ◽

Transcriptional Activation ◽

Network Formation ◽

Xenograft Model ◽

The Cancer Genome Atlas ◽

Sequencing Data ◽

Responder Cell

Abstract Microtubes (MTs) are cytoplasmic extensions of glioma cells serving as important cell communication structures while also promoting invasion and treatment resistance through network formation. MTs are abundant in chemoresistant gliomas, in particular glioblastomas, while they are uncommon in chemosensitive IDH mutated and 1p/19q co-deleted oligodendrogliomas. By performing a bioinformatics analysis on data from The Cancer Genome Atlas (TCGA) we identified the TGF-b pathway as being distinctly upregulated in glioblastomas compared to oligodendrogliomas, making this a signaling pathway potentially involved in MT formation. Based on patient-derived GBM stem cell line models we demonstrated that stimulation of TGF-b increased MT formation, while inhibition of TGF-b reduced MT formation. MT formation was verified by expression of GAP43 and nestin, which have previously been shown to be important structural proteins of MTs. Interestingly, we also observed a responder/non-responder relationship between GBM cell lines P3 and GG16/ GG6 regarding MT formation upon TGF-b stimulation. To determine downstream signaling mediators of the TGF-b pathway crucial for MT formation, we subsequently performed RNA sequencing of these cell lines. From the 34 initial candidates common to responders, but absent in non-responders, only 3 genes were left after filtering through TCGA data and in vivo RNA sequencing data of a GBM xenograft model derived from P3. Thrombospondin 1 (TSP1) emerged as the most interesting candidate as we have previously shown that transcription of this gene is activated by TGF-b/SMAD signaling and TSP1 also promotes invasiveness of GBM. TSP1 was upregulated by TGFB1 stimulation in responder cells and promoted MT formation. Transcriptional activation of TSP1 was absent in the non-responder cell line GG6 and could be reversed in the responder cell line P3 by TSP1 shRNAs in vitro and in vivo. Thus, TSP1 was experimentally verified as an important mediator of microtube formation downstream of TGF-b signaling.

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Abemaciclib in Combination with Copanlisib to Overcome Therapeutic Resistance in Mantle Cell Lymphoma

Blood ◽

10.1182/blood-2021-150942 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2253-2253

Author(s):

Yuxuan Che ◽

Yang Liu ◽

Yijing Li ◽

Joseph McIntosh ◽

Alexa A Jordan ◽

...

Keyword(s):

Cell Cycle ◽

Cell Line ◽

Cell Lines ◽

Research Funding ◽

Clinical Care ◽

Single Agent ◽

Xenograft Model ◽

Cancer Center ◽

Cell Viability Assay

Abstract Introduction Although novel therapeutic strategies including BTK and Bcl-2 inhibitors have dramatically improved the prognosis of MCL patients, resistance to these treatments is inevitable. We recently reported that the tumor suppressor gene CDKN2A were commonly deleted in ibrutinib-resistant tumors, leading to upregulation of CDK4/6 signaling. Among the other hallmarks are the mTOR/PI3K, Myc and OXPHOS pathways. Therefore, we attempt to exploit combinatory targeting of CDK4/6 and PI3K pathways to overcome therapy resistance using in vitro and PDX models. Methods Ibrutinib or venetoclax sensitive and resistant MCL cell lines were used in this preclinical study. 1x10 4 cells per well are seeded in 96-well plates and treated with abemaciclib monotherapy or in combination with copanlisib (PI3K inhibitor) in triplicate for 72h and then mixed with CellTiter-Glo Luminescent Cell viability Assay Reagent. For cell cycle assay, cells were seeded in 6 well plates and treated with vehicle or abemaciclib for 24h. Cells were fixed in 70% pre-cold ethanol and stained with propidium iodide. The cell cycle stages were quantified through the Novocyte Flow Cytometer. The molecular events at the protein level after treatment were determined by immunoblotting. For in vivo experiment, the combination of abemaciclib (25mg/kg, oral, daily) and copanlisib (5mg/kg, IP, three times a week) was assessed in Mino-venetoclax-resistant xenograft model. IC50 values were calculated using GraphPad Prism 8 for each cell line. Student's t-test was performed to compare the difference between vehicle and treated groups. Two-way analysis of variance (ANOVA) was conducted to analyze the tumor growth in vivo experiments. P values less than 0.05 were considered statistically significant. Results Our previous studies have identified a subset of MCL cells that were resistant to venetoclax (JeKo-1) or ibrutinib treatment (Maver-1 and Z-138). To overcome the resistance, we first treated MCL cell lines with abemaciclib and the result showed that abemaciclib as a single agent showed potent anti-MCL activity in a subset of MCL cell lines (IC 50 = 70-952 nM) including venetoclax sensitive- (Mino, Rec-1, Maver-1, and Z138) and primary resistant- MCL cells (JeKo-1). However, the cell lines Mino-ven-R and Rec-ven-R with acquired venetoclax resistance are highly resistant to abemaciclib treatment (IC 50 = 6.0 and 4.4 µM). PI3K/ATK pathway has been reported to be highly upregulated in Mino-ven-R and Rec-ven-R cells compared to their parental cells. To further increase the efficacy of the targeted therapy, we treated the resistant MCL cells with a combination of abemaciclib and copanlisib and the result showed synergistically enhanced cytotoxicity in ibrutinib or venetoclax-resistant MCL cell lines. Consistent with the role of CDK4/6 in cell cycle progression, inhibition of CDK4/6 with abemaciclib resulted in the cell cycle arrest at G1 phase in MCL cell lines. To validate whether abemaciclib in combination with copanlisib can overcome venetoclax resistance in vivo, we assessed the antitumor effect of abemaciclib in combination with copanlisib using a venetoclax-resistant xenograft models derived from Mino-ven-R cell line in immunodeficient NSG mice. As a result, abemaciclib (25 mg/kg, oral, daily), but not venetoclax (5 mg/kg, oral, daily) or copanlisib (5 mg/kg, IP, three times a week), significantly reduced tumor volume compared to the vehicle control (n = 5, p < 0.0001). Remarkably, the combination of abemaciclib and copanlisib also exhibited significantly in vivo synergistic efficacy compared with single-agent treatment (p<0.0001). Of note, the combination did not cause major decreases in body weight. Taken together, these results suggest that the combinatory therapy is effective in overcoming venetoclax resistance in MCL. Conclusions Combinatory treatment with abemaciclib and copanlisib may achieve clinical actionable efficacy through overcoming the venetoclax-resistance in MCL that may become an effective treatment regimen for refractory/relapsed MCL patients in the future. Disclosures Wang: Dava Oncology: Honoraria; Imedex: Honoraria; CStone: Consultancy; Hebei Cancer Prevention Federation: Honoraria; OMI: Honoraria; Chinese Medical Association: Honoraria; Newbridge Pharmaceuticals: Honoraria; Moffit Cancer Center: Honoraria; Bayer Healthcare: Consultancy; Kite Pharma: Consultancy, Honoraria, Research Funding; Clinical Care Options: Honoraria; Physicians Education Resources (PER): Honoraria; AstraZeneca: Consultancy, Honoraria, Research Funding; Mumbai Hematology Group: Honoraria; InnoCare: Consultancy, Research Funding; Epizyme: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Genentech: Consultancy; DTRM Biopharma (Cayman) Limited: Consultancy; Acerta Pharma: Consultancy, Honoraria, Research Funding; Scripps: Honoraria; BGICS: Honoraria; CAHON: Honoraria; BeiGene: Consultancy, Honoraria, Research Funding; Anticancer Association: Honoraria; Miltenyi Biomedicine GmbH: Consultancy, Honoraria; The First Afflicted Hospital of Zhejiang University: Honoraria; Juno: Consultancy, Research Funding; Loxo Oncology: Consultancy, Research Funding; Oncternal: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; VelosBio: Consultancy, Research Funding; BioInvent: Research Funding; Celgene: Research Funding; Lilly: Research Funding; Molecular Templates: Research Funding.

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Pre-Clinical Development of Adct-402, a Novel Pyrrolobenzodiazepine (PBD)-Based Antibody Drug Conjugate (ADC) Targeting CD19-Expressing B-Cell Malignancies

Blood ◽

10.1182/blood.v126.23.1564.1564 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1564-1564 ◽

Cited By ~ 7

Author(s):

Francesca Zammarchi ◽

David G. Williams ◽

Lauren Adams ◽

Karin Havenith ◽

Simon Chivers ◽

...

Keyword(s):

Single Dose ◽

B Cell ◽

Cell Lines ◽

Xenograft Model ◽

B Cell Malignancies ◽

Employment Equity ◽

Equity Ownership ◽

Anti Tumor Activity

Abstract Human CD19 antigen is a 95 kilodalton type I transmembrane glycoprotein belonging to the immunoglobulin superfamily (Wang, Wei, & Liu, 2012). The role of CD19, both in health and disease, is well studied, and the therapeutic efficacy and safety of CD19 modulation have been well defined over several decades (Scheuermann & Racila, 1995). In normal human tissue, expression of CD19 is limited to the various stages of B-cell development and differentiation (except plasma cells) and its expression is maintained on the majority of B-cell malignancies, including B-cell leukemia and non-Hodgkin lymphomas of B-cell origin. CD19 has rapid internalization kinetics and it is not shed into the circulation (Blanc et al., 2011; Gerber et al., 2009). All these features make CD19 an attractive target for the development of an ADC to treat B-cell malignancies. ADCT-402 is an ADC composed of a humanized antibody directed against human CD19, stochastically conjugated via a valine-alanine cleavable, maleimide linker to a PBD dimer cytotoxin. PBD dimers are highly efficient anticancer drugs that covalently bind in the minor groove of DNA and form cytotoxic DNA interstrand cross-links. The average drug to antibody ratio of ADCT-402 is 2.3 ± 0.3, as shown by hydrophobic interaction chromatography and reverse-phase HPLC. In vitro, ADCT-402 demonstrated potent cytotoxicity in a panel of human-derived cell lines of differing levels of CD19, while its potency was strongly reduced in CD19-negative cell lines. In vivo, ADCT-402 demonstrated dose-dependent anti-tumor activity in a subcutaneously implanted human Burkitt's lymphoma-derived Ramos xenograft model, where a single dose at 0.33 mg/kg induced significantly delayed tumor growth compared to the vehicle-treated mice and at 0.66 mg/kg and 1 mg/kg gave 4/10 and 10/10 tumor-free survivors, respectively. In the same model, ADCT-402 showed remarkably superior anti-tumor activity compared to both maytansinoid- and auristatin-based CD19-targeting ADCs, when they were tested at the same dose and schedule (1 mg/kg, single dose). Moreover, ADCT-402 mediated an impressive increase in survival compared to both vehicle-treated and isotype control ADC-treated mice in the disseminated Ramos xenograft model when tested as a single dose at 0.33 mg/kg or 1 mg/kg. For example, a single dose of ADCT-402 at 1 mg/kg resulted in 10/10 survivors at day 91, while there were 0/10 survivors at day 19 in the group of animals treated with either the vehicle control or with a single dose of the non-binding, control ADC at 1 mg/kg. In rat, a single dose of ADCT-402 at 2 mg/kg was well tolerated with no adverse signs or hematologic effects. Altogether, these data show the potent and specific anti-tumor activity of ADCT-402 against CD19-expressing B-cell malignancies, both in vitro and in vivo, and warrant further development of this ADC into the clinic. Disclosures Zammarchi: ADC Therapeutics: Employment. Williams:Spirogen/Medimmune: Employment. Adams:Spirogen/Medimmune: Employment, Equity Ownership. Havenith:ADC Therapeutics: Employment. Chivers:ADC Therapeutics: Employment. D'Hooge:Spirogen/Medimmune: Employment, Equity Ownership. Howard:ADCT Spirogen/Medimmune: Employment, Equity Ownership, Patents & Royalties. Hartley:ADCT Spirogen/Medimmune: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. van Berkel:ADC Therapeutics: Employment, Equity Ownership, Patents & Royalties.

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HDACi Targets IKZF1 Deletion High-Risk Acute Lymphoblastic Leukemia By Inducing IKZF1 Expression and Rescuing IKZF1 Function in Vitro and In Vivo

Blood ◽

10.1182/blood-2021-152926 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 514-514

Author(s):

Kangyu Huang ◽

Bingqing Tang ◽

Zihong Cai ◽

Xianjun He ◽

Qiuli Li ◽

...

Keyword(s):

High Risk ◽

Cell Line ◽

Cell Lines ◽

Preliminary Data ◽

Warburg Effect ◽

Selective Inhibitor ◽

Metabolic Reprogramming ◽

Dependent Manner ◽

Pdx Model

Abstract The IKZF1 gene codes the transcription factor IKAROS with key regulatory functions in lymphopoiesis. Recurrent focal IKZF1 deletions (IKZF1 del), which affects the coding regions of IKZF1, have been identified as poor outcome in 30-40% adult B-ALL. Loss of IKAROS confer stem cell-like phenotype, upregulation of self-renewal capacity and cell-adhesion molecules, and TKI resistance. However, there are not specifically therapeutic options for IKZF1 del ALL and current protocols could not abrogate the adverse effect of IKZF1 del.Considering that IKAROS acts as a key component of the nucleosome remodeling and deacetylation (NuRD) complex engaging in development and metabolism, we speculate that epigenetic drugs, such as HDACi, might play a potent role in IKZF1 del high-risk B-ALL. Firstly, B-ALL cell lines (IKZF1 del: MUTZ-5, MHH-CALL-4; IKZF1 wt: NALM6) and primary patient samples (n=10, 5 with IKZF1 del and 5 with IKZF1 wt) were treated with different HDACi, including valproic acid, vorinostat, romidepsin, RGFP966 and a novel HDAC-selective inhibitor tucidinostat. But noteworthily, only tucidinostat yielded specific and selective proliferation inhibition in IKZF1 del cell line(IC 50=1.377±0.05) and IKZF1 del patients samples (IC 50=2.318±0.07), compared with the effect on IKZF1 wt cells. Interestingly, tucidinostat induced remarked increase of mRNA and protein of IKZF1 expression in leukemia bulk and IKZF1 del single cell. Seahorse metabolic flux assay, lactate and ATP measurements was performed and revealed that tucidinostat treatment reduced glycolysis (P=0.0067), lactic acid (P＜0.0001) and ATP level (P＜0.0001) in IKZF1 del B-ALL cell lines. To verify metabolic change is depend on IKZF1 induction or not,dominant-negative Ikaros isoform 6 (DN-IK6), deletion of exons 4-7, was transfected into IKZF1 wt Nalm-6 cell line to negative regulate of IKZF1 wide-type expression. Overexpression of DN-IK6 in Nalm-6, increases sensitivity to tucidinostat, glycolytic capacity(p=0.05) and glycolytic reserve (p=0.012) also increases. While tucidinostat treating with the IK6-Nalm-6, tucidinostat would restore the transcriptional repressor function of the remaining wild-type IKZF1 allele and decrease glycolytic capacity(p=0.011) and glycolytic reserve(p=0.014). Notably, the metabolic rate-limiting enzymes HK2 and PKM2 were strongly repressed. These data indicate that tucidinostat reverses the metabolic reprogramming of glycolysis or Warburg effect in IKZF1 del B-ALL in an IKZF1-inducing dependent manner. For in vivo study, PDX model with immunodeficient NOD/SCID/IL2Rgnull mice were injected with heavily-treated refractory/relapsed IKZF1 del B-ALL patient samples (n=2) and treated with tucidinostat with different dosage of 5-12.5mg/kg/day. Administration of tucidinostat observed IKAROS expression trajectory and resulted in prolonged animal survival in IKZF1 del B-ALL PDX model（P＜0.0001). Secondary transplantation of ALL cells from tucidinostat or vehicle-treated (1 x10 6) recipients revealed significantly improved survival in tucidinostat -treated group (p= 0.0235). These results indicate that tucidinostat treatment might elimination leukemia-initiating cells.Additionally, to profile the IKZF1 del B-ALL chromatin accessibility changes after tucidinostat-treatment. We performed ATAC-seq and observed a clear increase in accessibility at TCA cycle related gene and decrease in accessibility at glycolysis related gene.Furthermore, tucidinostat, formerly known as chidamide, was added to an open-label, one-arm PDT-Ph-like-ALL trial targeting adult Ph-like ALL, which is characterized with high frequency in IKZF1 deletions (Clinicaltrials.gov. NCT03564470). Preliminary data of PDT-Ph-like-ALL indicate that tucidinostat was effective and well-tolerated, yielded promising response in IKZF1 del Ph-like ALL (ASH2018, poster 4011; EHA 2019, PF181). Collectively, our study demonstrates that the novel HDAC-selective inhibitor, tucidinostat, could specifically target IKZF1 del high-risk B-ALL, by restoring the IKZF1 expression, resulting in attenuation of proliferation, reverse the Warburg effect and improvement of the survival in PDX model and preliminary data in clinical trial. These findings provide mechanistic insights and a promising therapeutic strategy for IKZF1 haploinsufficiency alterations B-ALL patients. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Cytotoxicity of chitosan ultrafine nanoshuttles on MCF-7 cell line as surrogate model for breast cancer

Current Drug Delivery ◽

10.2174/1567201817666200719005440 ◽

2020 ◽

Vol 17 ◽

Author(s):

Tarek Faris ◽

Gamaleldin I. Harisa ◽

Fars K. Alanazi ◽

Mohamed M. Badran ◽

Afraa Mohammad Alotaibi ◽

...

Keyword(s):

Red Blood Cells ◽

Factorial Design ◽

Cell Line ◽

Cell Lines ◽

Blood Cells ◽

Sodium Tripolyphosphate ◽

Physicochemical Characterization ◽

Spherical Shape ◽

Mcf 7

Aim: This study aimed to explore an affordable technique for the fabrication of Chitosan Nanoshuttles (CSNS) at the ultrafine nanoscale less than 100 nm with improved physicochemical properties, and cytotoxicity on the MCF-7 cell line. Background: Despite several studies reported that the antitumor effect of CS and CSNS could achieve intracellular compartment target ability, no enough available about this issue and further studies are required to address this assumption. Objectives: The objective of the current study was to investigate the potential processing variables for the production of ultrafine CSNS (> 100 nm) using Box-Benhken Design factorial design (BBD). This was achieved through a study of the effects of processing factors, such as CS concentration, CS/TPP ratio, and pH of the CS solution, on PS, PDI, and ZP. Moreover, the obtained CSNS was evaluated for physicochemical characteristics, morphology Also, hemocompatibility, and cytotoxicity using Red Blood Cells (RBCs) and MCF-7 cell lines were investigated. Methods: Box-Benhken Design factorial design (BBD) was used in the analysis of different selected variables. The effects of CS concentration, sodium tripolyphosphate (TPP) ratio, and pH on particle size, Polydispersity Index (PDI), and Zeta Potential (ZP) were measured. Subsequently, the prepared CS nanoshuttles were exposed to stability studies, physicochemical characterization, hemocompatibility, and cytotoxicity using red blood cells and MCF-7 cell lines as surrogate models for in vivo study. Result: The present results revealed that the optimized CSNS have ultrafine nanosize, (78.3±0.22 nm), homogenous with PDI (0.131±0.11), and ZP (31.9±0.25 mV). Moreover, CSNS have a spherical shape, amorphous in structure, and physically stable. Also, CSNS has biological safety as indicated by a gentle effect on red blood cell hemolysis, besides, the obtained nanoshuttles decrease MCF-7 viability. Conclusion: The present findings concluded that the developed ultrafine CSNS has unique properties with enhanced cytotoxicity. thus promising for use in intracellular organelles drug delivery.

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