Daudi Cells Research Articles

Targeting Homologous Recombination in Lymphoid Malignancies: Evaluation of Four Small Molecule Inhibitors of RAD51

Genomic instability is recognized as a driver of tumorigenesis and cancer progression. Loss of tumor suppressors or activation of oncogenes can induce DNA damage stress, promoting genomic instability and creating dependencies upon key DNA repair pathways. These dependencies can be targeted therapeutically to induce synthetic lethality. The homologous recombination (HR) repair pathway is an attractive target. HR deficient cancers are hypersensitive to numerous anticancer drugs, and tumors will often induce expression of HR genes to promote drug resistance. RAD51 is a key component of the HR pathway. RAD51 forms nucleoprotein filaments at sites of DNA damage and replication fork stalls, mediating homologous DNA strand exchange to promote recombinational repair of breaks and damaged replication forks. We utilized four small molecule inhibitors of RAD51-mediated HR for evaluation of RAD51 as a potential therapeutic target. Compounds CYT-0851, CYT-0853, CYT-1027, and CYT-1127 were evaluated for anti-cancer activity in vitro and in vivo. To determine the impact of the small molecules on RAD51 and HR, all four were tested for effects on RAD51 focus formation and sister chromatid exchange (SCE) activity. All the compounds showed a reduction in SCE activity, however only CYT-0851 and CYT-0853 produced a measurable reduction in RAD51 foci. We have previously shown that that RAD51 inhibition leads to accumulation of DNA breaks, and ultimately cell death, in cells expressing the DNA mutator protein Activation Induced Cytidine deaminase (AICDA/AID). Cytotoxicity assays were performed in an AID+ (Daudi, Burkitt's Lymphoma) and AID- (WI-38, fibroblast) cell lines. All four compounds were preferentially active in AID+ cells with little to no cytotoxicity observed in the AID-negative WI-38 cell line. CYT-0853 was the most potent in the Daudi cell line with an EC50 of 8nM. All four compounds were orally bioavailable in all preclinical species tested but showed differences in pharmacokinetics. Preclinical cell line derived xenograft models of AID-high Burkitt's lymphoma (Daudi) and B-cell acute lymphoblastic leukemia (CCRF-SB) were used to determine the in vivo anti-tumor activity of the compounds in lymphoid cancer models. CYT-0851 and CYT-0853 both showed significant anti-tumor activity with tumor growth inhibition of greater than 50% in both models. Further analysis showed drug exposure with CYT-0851 was more consistent in the CDX models than CYT-0853. Overall, these data indicate that RAD51 and HR are attractive therapeutic targets for the treatment of lymphoid malignancies and that CYT-0851 is a viable clinical development candidate. Disclosures Day: Cyteir Therapeutics: Employment. Maclay:Cyteir Therapeutics: Employment. Cyr:Cyteir Therapeutics: Employment. Mills:Cyteir Therapeutics: Employment, Equity Ownership.

Preclinical Evaluation of Human Placental-Derived Allogeneic CD19 CAR-T Cells Against B Cell Malignancies

Celularity, Inc. is developing a CD19 CAR-T Cell therapy using an allogeneic platform derived from postpartum human placental cells. T cells isolated from placenta/ umbilical cord blood and genetically modified to express CD19 chimeric antigen receptor (CAR), termed Placental-derived (P-) CD19 CAR T cells, are in development for the treatment of B cell malignancies. Unlike adult peripheral blood mononuclear cell (PBMC)-derived T cells, P-T cells are mostly naïve (CD45RA+) and can be readily expanded while maintaining an earlier differentiation phenotype such as greater expression of naïve/ memory markers, lower expression of effector/ exhaustion markers, allowing for greater proliferative potential of these cells ex vivo. These cells are also known to have greater immune tolerance to HLA mismatch and display impaired allogeneic activation, contributing to lower incidences of severe graft-verse-host disease (GvHD) (Barker, et. al. Blood, 2001; Chen, et al. Biology of Blood and Marrow Transplantation, 2006), making them an attractive cell population for use as an allogeneic, adoptive cell therapy. A robust process for the isolation, transduction, and expansion of placental-derived T cells to generate "off-the-shelf" allogeneic P-CD19 CAR T cells was developed. Twenty-One day expanded, non-modified P-T cells (N=3) were compared to adult PBMCs for their allo-reactivity in a Xenogeneic GvHD model in NCG mice. P-T cells did not induce xeno-GvHD whereas PBMCs did, as evidenced by significant weight loss and death of all mice (N=5) by Day 28 post infusion. Despite expanded P-T cells demonstrating lack of in vivo GvHD, current manufacture of P-CD19 CAR T cells does include a CRISPR-mediated T-cell receptor a constant (TRAC) knockout (KO) step as an additional risk-mitigation strategy to circumvent any potential GvHD stemming from expression of endogenous T cell receptor. CD19 CAR transduction using a retrovirus provided by Sorrento Therapeutics, Inc., followed by TRAC knockout with CRISPR results in both high efficiency of CD19 CAR expression (~30% CD19 Fc+) and TCR KO (>96% CD3-/ TCR a/b-). In vitro, the functional activity of P-CD19 CAR-TRAC KO T cells against CD19+ Burkitt's Lymphoma (Daudi) and Acute lymphoblastic Leukemia (NALM6) cell lines was assessed in cytotoxicity and cytokine release assays. P-CD19 CAR T cells specifically lyse CD19+ Daudi/ Nalm6 targets in both 4-hour endpoint FACS and ACEA kinetic cytotoxicity assays, and in most cases at levels equivalent to or greater than PBMC-derived CD19 CAR T cells. When P-CD19 CAR T cells were co-cultured with CD19+ Daudi/ Nalm6 target cells for 24-hours, they secreted pro-inflammatory cytokines and effector proteins in an antigen-specific manner. In vivo, the anti-tumor activity of P-CD19 CAR T cells was assessed using a disseminated lymphoma xenograft model in NSG mice. Luciferase expressing Daudi cells (3×106) were intravenously (IV) injected on Day 0, followed by IV injection of P-CD19 CAR T cells (14×106) on Day 7. Bioluminescence Imaging (BLI) and survival were used as primary study endpoints. P- CD19 CAR T cells were well tolerated and safe. P-CD19 CAR T cells significantly reduced tumor burden, and improved survival. Four weeks after treatment, the vehicle group had a 100% mortality rate, while all animals from P-CD19 CAR T-treated group (N=5) remained alive without clinical symptoms including weight loss or changes in their fur. In summary, Celularity has defined a robust process for the generation and expansion of CD19 CAR T cells from human placenta. These cells exhibit potent anti-tumor activity both in vitro and in vivo with little evidence of acute GvHD induction, highlighting their potential as an allogeneic, adoptive cell therapeutic agent. Future in vivo GvHD studies will include assessment of both CD19 CAR and TRAC KO genetically modified P-T cells. Disclosures Karasiewicz: Celgene: Equity Ownership; Celularity, Inc.: Employment, Equity Ownership, Patents & Royalties: Patent Inventor. He:Celularity Inc: Employment. Ng:Celularity, Inc.: Employment. Tess:Celularity, Inc.: Employment. Ling:Celularity Inc: Employment. Kaufmann:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Zeldis:Sorrento Therapeutics Inc: Employment, Equity Ownership. Ji:Celularity, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Sorrento Therapeutics Inc: Employment, Equity Ownership, Patents & Royalties. Hariri:Celularity Inc: Employment. Zhang:Celularity Inc: Employment.

Development of CD38 CAR Engineered Human Placental Hematopoietic Stem Cell Derived Natural Killer Cells (PNK-CAR38) As Allogeneic Cancer Immunotherapy

Background: Natural killer (NK) cells exhibit innate anti-tumor activity owing to the expression of a multitude of activating receptors. Celularity has developed a GMP process for generating off-the-shelf and allogeneic human Placental Hematopoietic Stem Cell derived Natural Killer (PNK-007) cells. To develop a tumor targeted approach, we have engineered PNK cells to express Chimeric Antigen Receptor (CAR) against tumor antigen CD38. PNK cells expressing CD38 CAR (PNK-CAR38) have enhanced anti-tumor function against CD38+ lymphoma and multiple myeloma (MM) cell lines in pre-clinical studies and do not exhibit on-target off-tumor cytotoxicity against healthy CD38+ cells. Here we report the development of a CD38 CAR specific NK cell therapy. Methods: PNK-CAR38 cells were generated through transduction of human placental CD34+ cells with a retroviral vector carrying second generation anti-CD38 chimeric antigen receptor (CAR2-anti-CD38 A2; CD38scFv-CD28CD3ζ) followed by expansion and differentiation of transduced CD34+ cells into NK cells in presence of cytokines including thrombopoietin, SCF, Flt3 ligand, IL-7, IL-15 and IL-2. The anti-tumor activity of untransduced PNK and PNK-CAR38 cells was assessed against Lymphoma (Daudi and Raji) and Multiple Myeloma (Molp8, LP1 and OPM2) cell lines at various effector to target (E:T) ratios. PNK-CAR38 in vivo anti-tumor activity was assessed in a disseminated lymphoma xenograft model in NSG mice. Briefly, luciferase-expressing Daudi cells (3×106) were intravenously (IV) injected at Day0, followed by IV injection of PNK-CAR38 cells (20×106) at Day1 and Day3. Tumor burden was assessed weekly by Bioluminescence Imaging (BLI). Differences between the groups were calculated using two-tailed Student t-test. Results: Placental CD34+ cells were efficiently gene-modified using a retroviral vector and achieved a median of 64% (range 38% to 87%; n=11 donors) CD38 CAR expression in PNK cells at the end of 35-day culture. The median PNK-CAR38 cell yield achieved was 29,000-fold (range 7,800-48,000 fold; n=8) with NK purity >90% measured by CD3-CD56+ population. In vitro an overall improvement in anti-tumor response by PNK-CAR38 over PNK cells was observed. In a NK resistant lymphoma cell line model Daudi, PNK-CAR38 cells from various donors (n=8) elicited 72%±10% lysis versus 6%±3.5% lysis by PNK alone (p<0.01) at 10:1 E:T ratio. PNK-CAR38 elicited significant cytotoxicity 27%±9% versus 13%±5% by PNK cells (p<0.01) against Raji cells depending on NK cell donor. Using MM lines, at 10:1 E:T ratio, PNK-CAR38 cells elicited 33%±16% lysis versus 21%±12% by PNK (p<0.01) against LP1; 44%±6% versus 28%±3% by PNK (p<0.01) against OPM2; and 32%±6% versus 2%±0.5% by PNK (p<0.01) against MOLP8. Since CD38 is also expressed on normal immune and progenitor cells, PNK-CAR38 cells were co-cultured with healthy donor-derived activated T cells and CD34+CD38+ progenitor cells. No on-target off-tumor cytotoxicity against these cells was observed. In the disseminated Daudi xenograft NSG model, PNK-CAR38 significantly reduced tumor burden (approximately 50% BLI reduction) compared to vehicle control 10 days post treatment. Conclusions: The novel PNK-CAR38 demonstrated enhanced in vitro cytotoxic potential against lymphoma and MM lines and showed no on-target off-tumor cytotoxicity against healthy donor-derived cells in vitro. In vivo anti-tumor activity of PNK-CAR38 was shown in a lymphoma disseminated NSG model. Further development of PNK-CAR38 for hematological malignancies is warranted. Disclosures Somanchi: Celularity Inc: Employment. Guo:Celularity, Inc.: Employment. He:Celularity Inc: Employment. Mathur:Celularity Inc: Employment. Difiglia:Celularity Inc: Employment. Rana:Celularity Inc: Employment. Ling:Celularity Inc: Employment. Edinger:Celularity Inc: Employment. Kaufmann:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Zeldis:Sorrento Therapeutics Inc: Employment, Equity Ownership. Ji:Sorrento Therapeutics Inc: Employment, Equity Ownership, Patents & Royalties; Celularity, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Hariri:Celularity Inc: Employment. Zhang:Celularity Inc: Employment.

A Phase 2a Open-Label Study to Investigate Safety and Tolerability (including the MTD), Efficacy, Pharmacokinetics, Pharmacodynamics and Immunogenicity of MT-3724 in Combination with Lenalidomide in Subjects with Relapsed or Refractory B-Cell Non-Hodgkin Lymphoma

Introduction: Diffuse large B cell lymphoma (DLBCL) is the most common histologic subtype of Non-Hodgkin Lymphoma (NHL) accounting for approximately 30-40% of NHL cases. Approximately 40% of all newly diagnosed DLBCL patients are either refractory or relapsed following initial response to therapy and represent a population with high unmet need for new therapeutic strategies to achieve or regain disease remission. Because of the near ubiquity and persistence of CD20 expression in B-cell malignancies, there is strong rationale to develop compounds with novel mechanisms of action targeting CD20. However, CD20's non-internalizing nature has, to date, leveraged only cytotoxic mechanisms such as antibody-dependent cellular cytotoxicity to which resistance can arise, and has impeded the development of agents that internalize a cytotoxic payload. MT-3724 is a novel engineered toxin body designed to overcome this limitation by combining the specific target selectivity of a single chain variable fragment with the lethality of a genetically fused Shiga-like toxin A subunit that facilitates both internalization and cell killing by inactivating ribosomal protein synthesis. MT-3724 has been shown to specifically bind and kill CD20+ malignant human B-cells in vitro (IC50 <1 nM) and in CB17 SCID and PDX mice (Rajagopalan 2016; Huang 2018). Given MT-3724's unique ability to kill CD20+ cells by ribosomal inhibition, combining MT-3724 with standard cytotoxic chemotherapy and/or immune modulatory agents may provide enhanced benefit in NHL treatment algorithms. To evaluate this possibility, MT-3724 was tested in combination with Lenalidomide (LEN) to assess additive, synergistic or antagonistic effects in CD20-positive Daudi cell lines. This combination significantly (Ki = 0.40) potentiated cytotoxicity. Based on these results and the known safety profile of MT-3724, LEN was chosen as a combination agent to maximize anti-tumor effects in patients with B-cell NHL while minimizing overlapping toxicity risks. Study Design and Methods: MT-3724 is being evaluated in this Phase 2a study (NCT03645395) in combination with LEN in adult patients with histologically confirmed, relapsed or refractory NHL. The primary objective of this study is to determine the safety and tolerability, including the maximum tolerated dose (MTD), of MT-3724 + LEN. Secondary objectives include pharmacokinetics (PK), pharmacodynamics (PD), immunogenicity and tumor response in subjects treated with MT-3724 + LEN. The study will be conducted in two parts. Part 1 will include MT-3724 dose escalation according to the modified 3+3 design to identify the MTD of MT-3724 in combination with standard doses of LEN and will include up to 24 subjects with histologically confirmed NHL. Part 2 is designed to confirm the safety and tolerability of MT-3724 + LEN in the MTD Expansion Cohort, where the dose declared as MTD of MT-3724 in Part 1 would be given in combination with LEN in up to 40 subjects with CD20+ DLBCL. In addition, the PK, PD, immunogenicity and tumor response of MT-3724 + LEN will be evaluated in Part 2. Eligible subjects will have histologically confirmed, relapsed or refractory CD20+ B-cell NHL that, in the investigator's opinion, could benefit from MT-3724 + LEN therapy. Patients must have received all approved therapies for NHL that are applicable for the patient in the opinion of the treating physician and must have measurable disease by Lugano Classification for NHL. Subjects that are refractory to treatment are eligible, as well as patients who have progressed following CAR T-cell therapy, autologous- or allogeneic stem cell transplant. Serum rituximab level must be negative (<500 ng/mL) at screening. Subjects will receive MT-3724 IV infusions over 1 hour on Days 1, 3, 5, 8, 10 and 12 and will receive LEN on days 1-21 of each 28-day treatment cycle during cycles 1 and 2. Thereafter, MT-3724 will be administered weekly on Days 1, 8, 15, and 22 of each cycle with continued LEN dosing on Days 1-21 for cycles 3 to 6. The study is currently ongoing. Eligible subjects will be identified and treated through competitive enrollment at multiple study centers in North America. Disclosures Burnett: Molecular Templates, Inc.: Employment. Gordon:Abbott: Equity Ownership; Abbvie: Equity Ownership. Strack:Molecular Templates, Inc.: Employment. Lehner:Molecular Templates, Inc.: Employment. Higgins:Molecular Templates, Inc.: Employment, Equity Ownership.

Artesunate activates the ATF4-CHOP-CHAC1 pathway and affects ferroptosis in Burkitt's Lymphoma

Currently, there is no effective treatment for Burkitt's lymphoma in patients aged above 60 years, and thus research on effective treatment options for Burkitt's lymphoma has been gaining increasing attention. Artesunate has been identified as a novel effective growth suppressor in Burkitt's lymphoma. Here, we utilized molecular biology, transcriptome analysis, and other techniques to study artesunate-induced death of the Burkitt's lymphoma cells DAUDI and CA-46, the effect of artesunate on gene expression in DAUDI and CA-46 cells, and the effect of artesunate-induced ATF4-CHOP-CHAC1 pathway on ferroptosis. We also studied the inhibitory effects and ferroptosis induction of artesunate on CA-46 cells in mouse xenografts. Results showed that artesunate induced ferroptosis in DAUDI and CA-46 cells, as evidenced by the protective effect of liproxstatin-1, ferrostatin-1, and desferoxamine, resulting in an endoplasmic reticulum stress response, activation of the ATF4-CHOP-CHAC1 pathway enhanced ferroptosis in DAUDI and CA-46 cells. A mouse-transplanted tumor model showed that artesunate can inhibit the proliferation and induce ferroptosis of CA-46 cells in vivo. This study provides a novel perspective for the development of drugs against different types of Burkitt's lymphomas.

A strategy to protect off-the-shelf cell therapy products using virus-specific T-cells engineered to eliminate alloreactive T-cells

BackgroundThe use of “off-the-shelf” cellular therapy products derived from healthy donors addresses many of the challenges associated with customized cell products. However, the potential of allogeneic cell products to produce graft-versus-host disease (GVHD), and their likely rejection by host alloreactive T-cells are major barriers to their clinical safety and efficacy. We have developed a molecule that when expressed in T-cells, can eliminate alloreactive T-cells and hence can be used to protect cell therapy products from allospecific rejection. Further, expression of this molecule in virus-specific T-cells (VSTs) should virtually eliminate the potential for recipients to develop GVHD.MethodsTo generate a molecule that can mediate killing of cognate alloreactive T-cells, we fused beta-2 microglobulin (B2M), a universal component of all human leukocyte antigen (HLA) class I molecules, to the cytolytic endodomain of the T cell receptor ζ chain, to create a chimeric HLA accessory receptor (CHAR). To determine if CHAR-modified human VSTs could eliminate alloreactive T-cells, we co-cultured them with allogeneic peripheral blood mononuclear cells (PBMC), and assessed proliferation of PBMC-derived alloreactive T-cells and the survival of CHAR-modified VSTs by flow cytometry.ResultsThe CHAR was able to transport HLA molecules to the cell surface of Daudi cells, that lack HLA class I expression due to defective B2M expression, illustrating its ability to complex with human HLA class I molecules. Furthermore, VSTs expressing CHAR were protected from allospecific elimination in co-cultures with allogeneic PBMCs compared to unmodified VSTs, and mediated killing of alloreactive T-cells. Unexpectedly, CHAR-modified VSTs eliminated not only alloreactive HLA class I restricted CD8 T-cells, but also alloreactive CD4 T-cells. This beneficial effect resulted from non-specific elimination of activated T-cells. Of note, we confirmed that CHAR-modified VSTs did not affect pathogen-specific T-cells which are essential for protective immunity.ConclusionsHuman T-cells can be genetically modified to eliminate alloreactive T-cells, providing a unique strategy to protect off-the-shelf cell therapy products. Allogeneic cell therapies have already proved effective in treating viral infections in the stem cell transplant setting, and have potential in other fields such as regenerative medicine. A strategy to prevent allograft rejection would greatly increase their efficacy and commercial viability.

The Anticancer and Anti-inflammatory Effects of Centaurea solstitialis Extract on Human Cancer Cell Lines.

Natural products originating from plants have been used for many years in the treatment of various diseases, including cancer. Centaurea solstitialis subsp. solstitialis is used in Turkish folk medicine. This study was the first to determine the in vitro biological effects of ethanolic extract from the flowering parts of C. solstitialis L. subsp. solstitialis collected from Muğla Province. The cytotoxic effect was evaluated against Daudi, A549, and HeLa cancer cells and one normal BEAS-2B cell line using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- dipenyltetrazolium bromide) assay. Flow cytometric analysis and the caspase-3 activity assay were performed to detect apoptotic cell death. Angiogenic factor [vascular endothelial growth factor (VEGF)] secretion and the release of interleukin (IL)-1α, IL-6, and tumor necrosis factor (TNF)-α by cells treated with the extract were measured using enzyme-linked immunosorbent assay. The extract exhibited cytotoxic effects against all the cancer cell lines used but HeLa and Daudi were the most sensitive cells, with IC50 values of 63.18 μg/mL and 69.27 μg/mL, respectively. Selective cytotoxicity was observed between the HeLa and normal BEAS-2B cell lines. The extract arrested the cell cycle at the S and G2 phases. In addition, apoptotic cell death was detected in HeLa and A549 cells. Moreover, the plant extract caused a significant decrease in VEGF secretion in A549 cells and a fluctuation in IL-1α, IL-6, and TNF-α secretion in A549 and Daudi cells. These observations suggest that the flowering parts of C. solstitialis may be a potential source in the development of natural drugs for the treatment of cancer and modulation of cytokine secretion.

T cell cytotoxicity toward hematologic malignancy via B7-H3 targeting.

T cells are important effectors in anti-tumor immunity, and aberrant expression of B7 family members may contribute to tumor evasion. In this study, we analyzed expression of costimulatory molecules on human hematologic tumor cells and explored whether B7-H3, a member of the B7 superfamily, is an effective target for T cell mediated cytotoxicity toward hematologic malignancy. We investigated the bispecific antibody anti-CD3 × anti-B7-H3 (B7-H3Bi-Ab) for its ability to redirect T cells to target B7-H3 positive hematologic tumors, including Thp-1, K562, Daudi cells and a primary culture. The capacity of T cells armed with B7-H3Bi-Ab to kill hematologic tumors was evaluated by lactate dehydrogenase assay, flow cytometry, ELISA, and luciferase quantitative assay at an effector/target ratio of 5:1. Compared with unarmed T cells, B7-H3Bi-Ab-armed T cells exhibited significant cytotoxicity toward hematological tumor cells. Moreover, B7-H3Bi-Ab-armed T cells secreted more IFN-γ, TNF-α, IL-2, and Granzyme B and expressed higher levels of activating marker CD69 compared to unarmed T cells. In conclusion, B7-H3Bi-Ab enhances the ability of T cells to kill hematologic tumor cells, and B7-H3 may serve as a novel target for immunotherapy against hematologic malignancy.

Abstract 876: CD40-induced growth inhibition of Burkitt lymphoma: A possible role for NADPH oxidase by upregulation of p67phox

Abstract CD40 stimulation together with IL4 has been shown to promote growth of normal B cells in vitro. In contrast to this, CD40 stimulation in several B cell lymphoma cell lines has been demonstrated to be growth inhibitory. Though the mechanism is not completely known, CD40-stimulated cells exhibit increased levels of reactive oxygen species (ROS). In B lymphocytes, a major source of ROS production is the NADPH oxidase (NOX) that is comprised of a membrane-bound Nox2 catalytic subunit and four cytosolic regulatory components, with the p67phox cytosolic protein being the activator factor. Upon stimulation, by either cross linking of the B-cell receptor or following treatment with PMA, the active NADPH oxidase generates superoxide. We have investigated the role of NADPH oxidase in CD40-mediated growth inhibition in Burkitt lymphoma (BL) cells. For this study, we first evaluated the effect of CD40 stimulation on the expression of Nox2 and p67phox in BL cells that demonstrate different levels of endogenous Nox2 and p67phox. Accordingly, Daudi and Raji cell lines, with endogenously high Nox2 and low p67phox, NAMALWA cells which have high Nox2 and no p67phox expression, and Ramos cells that contain no Nox2 or p67phox were analyzed. Treatment with anti-CD40 or CD40 ligand (CD40L) for 24 h induced up-regulation of p67phox in Daudi, Ramos, and NAMALWA cells, but not in the Raji line. No significant change in the expression of Nox2 was observed. Additionally, Daudi, Ramos and NAMALWA cells showed higher production of ROS after CD40 stimulation and activation with anti-IgM or PMA, in a chemiluminescence assay. To determine whether the change in ROS level was related to the upregulation of p67phox, Daudi cells that express either stable p67phox shRNA or control shRNA were generated. Treatment with CD40L for 24 h demonstrated significant ROS production only in control shRNA and not in the p67shRNA cells, confirming that the ROS production is NOX2-p67phox mediated. This enhanced ROS production was observed following treatment with CD40L alone and was enhanced further in conjunction with anti-IgM or PMA. However, cells with the p67phox shRNA showed detectable level of ROS only after activation with anti IgM, albeit to much lower levels than the control. Co-treatment of Daudi cells with anti-CD40 or CD40L and IL4 further increased the expression of p67phox. Although the basal level of ROS in the cells was unchanged upon co-treatment, the ROS generated following stimulation with anti-IgM or PMA was significantly enhanced. Preliminary MTT studies confirmed the growth inhibition of CD40-stimulated Daudi cells but not of Raji cells, suggesting a role for p67phox-dependent, ROS-related inhibition of lymphoma cell proliferation. Experiments are currently ongoing to delineate further the role of CD40 stimulation and Nox2-p67phox mediated ROS in affecting the growth of Burkitt lymphomas and signaling pathways downstream of CD40. Citation Format: Mazal Iris Dahan, Smitha Antony, Agnes Juhasz, Guojian Jiang, Mariam Konate, Jiamo Lu, Jennifer Meitzler, YongZhong Wu, Krishnendu Roy, James H. Doroshow. CD40-induced growth inhibition of Burkitt lymphoma: A possible role for NADPH oxidase by upregulation of p67phox [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 876.

Abstract 1741: Identification of microsatellite instability in hematologic malignant cell lines

Abstract Defective DNA repair machinery which is a consequence of chromosomal or molecular alterations is associated with variety of diseases including solid tumors. Cytogenetic analysis is an important tool in diagnosis and treatment of hematologic malignancies. Our aim is to understand the role of microsatellite instability in initiation and progression of blood cancers. Here we hypothesize that leukemogenic cells may carry defective repair mechanisms that contribute to other genomic instabilities. In order to explore the possible role of microsatellite instability (MSI) in leukemia and lymphoma, eight different DNA repair genes involved in mismatch repair (MMR) and direct reversal of damage (DRD) were analyzed in K562 (CML), Molt-4 (T-ALL) and Daudi (Burkitt’s lymphoma) cell lines. Fragment analysis was carried out using novel primers considering repeat motifs enriched with (A)n, (CA)n and (AT)n for instability. Almost all the cell lines showed moderate to high level of microsatellite instability and exhibited a shift in selected repeat regions indicating possible loss (66.7%) (MSH2, MSH4, MSH6, MLH1, MLH3, PMS1) or gain (25%) (MSH3, MGMT) of nucleotide repeats as compared to normal DNA. The K562, Philadelphia negative CML cell line appeared to be microsatellite stable (MSS) for genes MSH2 and MLH1. Treatment induced microsatellite instability is known in leukemic patient cells as also seen in Molt-4 cells. Our present and previous observations suggest that MSI is a non-random event in leukemia/lymphoma which needs confirmatory future studies in newly diagnosed untreated patient samples. Considering the fact that both MSI and MSS represents two distinct molecular pathways of carcinogenesis, assessment of MSI, if added as distinct diagnostic criteria, can possibly refine early detection and help in identifying novel targets in management of hematologic malignancies. Citation Format: Priyanjali Bhattacharya, Naveed Anjum Chikan, Arati Sharma, Dhimant Desai, Charyguly Annageldiyev, Pinaki Patel, Trupti N. Patel. Identification of microsatellite instability in hematologic malignant cell lines [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 1741.

Abstract 3265: NKTR-255, a polymer-conjugated IL-15 enhances anti-tumor NK cell responses and synergizes with monoclonal antibodies to provide long-term survival in human lymphoma model

Abstract Background: IL-15 is a cytokine that activates and provides survival benefit to NK cells. Exploiting the therapeutic value of native IL-15 has been challenging due to its unfavorable pharmacokinetic properties and tolerability. NKTR-255 is a polymer-conjugated human IL-15 that retains binding affinity to the alpha subunit of IL-15 receptor and exhibits reduced clearance to thereby provide a sustained pharmacodynamics response. NKTR-255 has potential for providing an enhanced immunotherapeutic effect when combined with monoclonal antibodies that mediate tumor killing by antibody dependent cellular cytotoxicity (ADCC). Here we investigate the pharmacological properties of NKTR-255 on NK cells and the therapeutic effect of NKTR-255 when combined with tumor-directed monoclonal antibodies in a B cell lymphoma model. Methods: KHYG-1 cells (human NK cell line) were used to measure phosphorylated STAT5 (pSTAT5) and cell proliferation. Human PBMCs were stimulated with NKTR-255 and/or daratumumab for in vitro NK cell characterization. In the cytotoxic assay, mice received single IV doses of 0.3 mg/kg of NKTR-255 and splenic NK cells were co-cultured with YAC-1 cells (mouse T lymphoma cell line) to measure cytotoxic function. In the Daudi lymphoma model, 1x107 Daudi cells were inoculated IV on Day 0. NKTR-255 (0.03, 0.1 or 0.3 mg/kg SC) was administered on Days 14, 21 and 28 and antibody treatment was administered on Day 14 (daratumumab 0.5 mg/kg IP) or on Days 14 and 17 (rituximab 40 mg/kg IP). Survival rate was determined by onset of hindlimb paralysis as a surrogate parameter. Results: NKTR-255 dose-dependently induced pSTAT5 and proliferation in KHYG-1 cells (EC50 values for pSTAT5: 0.2 ng/ml, proliferation: 5 ng/ml). NKTR-255 also resulted in enhanced pSTAT5 and NKG2D surface expression on human primary NK cells. In addition NKTR-255 increased NK cell degranulation in co-culture experiments with U266 cells (myeloma cell line) with/without daratumumab (anti-human CD38 antibody), as determined by enhanced CD107a surface expression. In vivo pretreatment with NKTR-255 resulted in sustained cytotoxic function of NK cells in both ex vivo and in vivo studies. Finally, NKTR-255 synergistically provided long-term survival benefit in a dose-dependent manner when administered with daratumumab or rituximab (anti-human CD20 antibody) in the Daudi B cell lymphoma model. Conclusions: NKTR-255 is an immune stimulator of NK cells that provides a dose-dependent effect in the proliferation and activation of NK cells. This property of NKTR-255, when administered with daratumumab or rituximab, translates into enhanced therapeutic efficacies of the antibodies in a B cell lymphoma model. These results indicate that combining NKTR-255 with a tumor-directed antibody having an ADCC mechanism can provide a synergistic effect for treating cancers. Citation Format: Takahiro Miyazaki, Saul Kivimäe, Rhoneil Pena, Peiwen Kuo, Marlene Hennessy, Murali Addepalli, Neha Dixit, Wildaliz Nieves, Sara Sheibani, Mekhala Maiti, Laurie VanderVeen, Joanna Wilczek, Loui Madakamutil, Jonathan Zalevsky. NKTR-255, a polymer-conjugated IL-15 enhances anti-tumor NK cell responses and synergizes with monoclonal antibodies to provide long-term survival in human lymphoma model [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 3265.

Evaluation of <i>in vitro</i> immunomodulatory properties of biologically active substances and drugs

A new combined method for screening of immunomodulatory properties of drugs was developed, including: expression of CD80, HLA-DR, CD32, CD205 and CD197, interleukin-12 production and apoptosis by DCs; production of reactive oxygen species and evaluation of phagocytosis by neutrophils; expression of CD69 and production of tumor necrosis factor-α by Jurkat-tat cells; and CD80 and HLA-DR expression, viability and apoptosis using the Daudi cell line.

The role of platelet microparticles in the production of antibodies from B lymphocytes against HLA-DR antigen in vitro

Background: Platelets can activate B cells and stimulate them for the production of antibodies. Since platelet microparticles (PMPs) originate from platelets, they may have the same virtue. In the present study, the effect of PMPs was investigated on the production of human leukocyte antigen (HLA)-specific antibody from B cells in vitro. Materials and Methods: In this experimental study, HLA-DR antigen was solubilized from the immortalized B lymphocytes (Daudi cell line) and purified using the affinity chromatography. Antigen properties were determined by the ELISA technique. PMPs were isolated from platelet concentrate bags by centrifugation. Fresh blood products were prepared from the Innovation Center of Iranian Blood Transfusion Organization (IBTO) and B lymphocytes were purified by the MACS method. B cells were exposed with PMPs and HLA-DR antigens in the culture medium. On the third day of culture, the culture supernatant was examined in terms of antibody production using the ELISA test. The results were analyzed using paired sample T-test and P-value <0.05 was considered statistically significant. Results: The specificity of the purified HLA-DR antigen was confirmed using the anti-HLA-DR antibody and ELISA technique in the presence of appropriate controls. The results showed that PMPs could stimulate the production of antibodies from B cells. The difference between the case and control was significant (P-value=0.001). Although total immunoglobulin (IgG) was higher in HLA-DR-treated wells, HLA-DR-specific antibodies were not identified by ELISA technique. Conclusion: PMPs have the capability to induce IgG antibodies from B cells. In order to ensure the production of specific antibodies, further testing is required with high sensitivity.

Role of platelet-derived microparticles in transfer of the chemokine receptor CXCR4 to CXCR4-negative cells.

Background: Membrane-derived microparticles (PMPs) are produced from platelets during activation, storage, and apoptosis. PMP can transfer some adhesion molecules such as CXCR4 to CXCR4-negative cells. In this study, the ability of PMPs to deliver CXCR4 molecule to CXCR4-null targets (Daudi, K562 and U937 cell line) was evaluated and the different concentrations of PMPs were examined to transfer CXCR4. Methods: In this experimental study, PMPs were prepared using serial centrifugations. After confirmation of PMP with flow cytometry, PMP concentration was evaluated using the Bradford method. CXCR4-negative cell lines (1×105 cells/ml) were cultured in RPMI1640 with 10% FBS and 1% antibiotic. PMPs in 7 different concentrations were added to cell culture plates and incubated for 1 hour at 37ºc and 5% CO2. The presence of CXCR4 on cells was analyzed by flowcytometry. Results: In this study, characterization of PMPs and cell lines were done by flow cytometry. Then, the PMPs’ ability to transfer CXCR4 to null cells (Daudi, K562 and U937 cell lines) was evaluated in 7 concentrations (10, 20, 50,125, 250, 500, 1000 μg/mL); incubation lasted for 1 hour. The best result of transferring CXCR4 by PMP was done in the concentration of 250µg/mL. Conclusion: PMPs in different concentrations can transfer CXCR4 to target cells. Also, the increase of PMPs concentration up to 250µg/mL can increase the CXCR4 presence on null cells.

Efficacy and mechanism of action of NKTR-255, a polymer-conjugated IL-15 receptor agonist in hematological tumors.

e19512 Background: NKTR-255 is a polymer-conjugated IL-15 receptor agonist that accesses the IL-15 signaling pathway, and enhances the function and numbers of natural killer (NK) cells and CD8 memory T cell populations. Many hematological malignancies are treated with targeted antibodies that utilize effector mechanisms to recruit NK cells and macrophages. Most patients with hematological malignancies experience a progressive decline of NK cell immunity, a phenomenon termed as an “immunological desert”. We evaluated the hypothesis that a combination of NKTR-255 with a targeted antibody such as an anti-CD38 monoclonal antibody (mAb) or an anti-CD20 mAb, when evaluated in a preclinical hematological tumor model, would provide enhanced efficacy. Methods: Human PBMCs were stimulated with NKTR-255 and/or daratumumab for in vitro NK cell characterization. Using a B cell lymphoma model, SCID mice were inoculated i.v. with 107 Daudi cells and treated with a single dose of daratumumab or rituximab two weeks after tumor cell injection. NKTR-255 was administered s.c. concurrently on a q7dx3 dosing schedule. Survival of treated animals was measured using hind limb paralysis and morbidity as surrogate endpoints. Tumor cell depletion and phenotyping of immune cells in bone marrow were conducted by flow cytometry. Results: NKTR-255, when combined with daratumumab, enhanced NK cell degranulation and decreased NK cell death, leading to enhanced antibody-dependent cellular cytotoxicity (ADCC) activity for the daratumumab - NKTR-255 combination over the single agents. NKTR-255 demonstrated synergistic activity and provided a long-term survival benefit when combined with daratumumab or rituximab in the CD38 and CD20-expressing lymphoma mouse models. Mechanistic studies conducted in tumor-bearing mice treated with a combination of NKTR-255 and daratumumab showed effective clearance of lymphoma cells from the bone marrow and increased expansion of effector NK cells and other immune cells. Depletion experiments confirmed the role of NK, CD8 and other monocyte lineage immune cells in the observed anti-myeloma activity. Conclusions: These studies indicate that administration of NKTR-255, when combined with a monoclonal antibody such as daratumumab and rituximab, significantly increases NK cell numbers and activates their tumor killing capacity by virtue of an enhanced ADCC effect. Combinatorial approaches of NKTR-255 with these targeting agents could lead to novel and effective clinical applications in hematology.

Daratumumab-225Actinium conjugate demonstrates greatly enhanced antitumor activity against experimental multiple myeloma tumors

ABSTRACT Daratumumab is an anti-CD38 directed monoclonal antibody approved for the treatment of multiple myeloma (MM) and functions primarily via Fc-mediated effector mechanisms such as complement-dependent cytotoxicity (CDC), antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cellular phagocytosis, and T-cell activation. However, not all patients respond to daratumumab therapy and management of MM remains challenging. Radioimmunotherapy with alpha particle-emitting radionuclides represents a promising approach to significantly enhance the potency of therapeutic antibodies in cancer treatment. Here we report the results of mechanistic and feasibility studies using daratumumab radiolabeled with an alpha-emitter 225Actinium for therapy of MM. CD38-positivelymphoma Daudi cell line and MM cell lines KMS-28BM and KMS-28PE were treated in vitro with 225Ac-daratumumab. 225Ac-daratumumab Fc-functional properties were assessed with C1q binding and ADCC assays. The pharmacokinetics and tumor uptake of 111In-daratumumab in Daudi tumor-bearing severe combined immunodeficiency (SCID) mice were measured with microSPECT/CT. The therapeutic effects of 225Ac-daratumumab on Daudi and KSM28BM tumors in mice and treatment side effects were evaluated for 50 days posttreatment. The safety of 225Ac-labeled antimurine CD38 mAb in immunocompetent mice was also evaluated. 225Ac-daratumumab efficiently and specifically killed CD38-positive tumor cells in vitro, while its complement binding and ADCC functions remained unaltered. MicroSPECT/CT imaging demonstrated fast clearance of the radiolabeled daratumumab from the circulation and tissues, but prolonged retention in the tumor up to 10 days. Therapy and safety experiments with 225Ac-daratumumab showed a significant increase in the antitumor potency in comparison to naked antibody without any significant side effects. Our results highlight the potential of targeting alpha-emitters to tumors as a therapeutic approach and suggest that 225Ac-daratumumab may be a promising therapeutic strategy for the treatment of hematologic malignancies.

Determination of anticancer effects of Urospermum picroides against human cancer cell lines

Plants continue to be a good source for developing effective anticancer agents. In this study, in vitro various biological effects of crude ethanolic extract from flowering parts of Urospermum picroides collected from the Muğla province of Turkey were investigated for the first time. Daudi, A549 and HeLa cancer cell lines and BEAS-2B normal cell line were used to identify the cytotoxic effect of the extract using MTT assay. The effect of the extract on cell cycle progression was detected by flow cytometric analysis. The level of VEGF, IL-1α, IL-6 and TNF-α secretion in the cells treated with the extract were measured using ELISA The extract caused a higher cytotoxic effect on Daudi cells with an IC50 value of 85.64 µg/mL than the other cells tested. The IC50 values in HeLa and A549 cells were determined to be 135.35 and 234.8 µg/ mL, respectively. The selective cytotoxicity was considered between Daudi and BEAS-2B (109.80 µg/mL) cell lines. In addition, the effect of the extract on cell cycle progression changes according to cell line used. Moreover, the extract decreased the level of secreted VEGF in treated A549 cells by 31%. In addition, the extract resulted in a significant decrease in the secretion of IL-1α, IL-6 and TNF-α cytokines in A549 and Daudi cells compared to the untreated cells. These findings suggest that the flowering parts of U. picroides may be a potential source for anticancer agents.

Design, synthesis and evaluation of novel 7H-pyrrolo[2,3-d]pyrimidin-4-amine derivatives as potent, selective and reversible Bruton's tyrosine kinase (BTK) inhibitors for the treatment of rheumatoid arthritis

A series of 7H-pyrrolo[2,3-d]pyrimidine derivatives were designed and synthesized as reversible BTK inhibitors, and evaluated their kinase selectivity, anti-proliferation against the B-cell lymphoma cell lines (Ramos, Jeko-1) and cell line BTK enhanced (Daudi) in vitro. Among them, compound 28a exhibited the most excellent potency (IC50 = 3.0 nM against BTK enzyme, 8.52 μM, 11.10 μM and 7.04 μM against Ramos, Jeko-1, Daudi cells, respectively), good kinase selectivity and inhibited BTK Y223 auto-phosphorylation and PLCγ2 Tyr1217 phosphorylation. Importantly, 28a showed efficacy anti-arthritic effect on collagen-induced arthritis (CIA) model in vivo. 28a 60 mg/kg dose level once a day group displayed markedly reduced joint damage and cellular infiltration without any bone and cartilage morphology change.

Targeting Burkitt lymphoma with a tumor cell–specific heptamethine carbocyanine‐cisplatin conjugate

BackgroundBurkitt lymphoma is a fast‐growing mature B cell malignancy, whose genetic hallmark is translocation and activation of the c‐myc gene. Prompt multiagent immunochemotherapy regimens can have favorable outcomes, but prognosis is poor in refractory or relapsed disease. We previously identified a novel family of near‐infrared heptamethine carbocyanine fluorescent dyes (HMCD or DZ) with tumor‐homing properties via organic anion‐transporting peptides. These membrane carriers have uptake in tumor cells but not normal cells in cell culture, mouse and dog tumor models, patient‐derived xenografts, and perfused kidney cancers in human patients.MethodsHere we report the cytotoxic effects of a synthesized conjugate of DZ with cisplatin (CIS) on B cell lymphoma CA46, Daudi, Namalwa, Raji, and Ramos cell lines in cell culture and in xenograft tumor formation. Impaired mitochondrial membrane permeability was examined as the mechanism of DZ‐CIS–induced lymphoma cell death.ResultsThe new conjugate, DZ‐CIS, is cytotoxic against Burkitt lymphoma cell lines and tumor models. DZ‐CIS retains tumor‐homing properties to mitochondrial and lysosomal compartments, does not accumulate in normal cells and tissues, and has no nephrotoxicity in mice. DZ‐CIS accumulated in Burkitt lymphoma cells and tumors induces apoptosis and retards tumor cell growth in culture and xenograft tumor growth in mice.ConclusionDZ‐CIS downregulated c‐myc and overcame CIS resistance in myc‐driven TP53‐mutated aggressive B cell Burkitt lymphoma. We propose that DZ‐CIS could be used to treat relapsed/refractory aggressive Burkitt lymphomas.

Highly Effective Treatment of CD38 Positive Experimental Lymphoma with 225Actinium-Daratumumab

Daratumumab is a human cytolytic antibody specific for CD38 that is used clinically for treatment of patients with multiple myeloma (MM). Current therapeutic regimens require multiple injections over months of treatment, therefor increasing the potency of daratumumab to shorten the length of treatment would be very advantageous. 225Ac is an α-particle emitting radionuclide that has potent cytotoxic activities over relatively short distances, allowing for precise targeting of a lethal dose of radiation. Previously we have established that labeling daratumumab with 225Ac increased more than 10-fold its ability to kill MM cell lines in vitro. In this study we evaluated the therapeutic potential of 225Ac-daratumumab by treating mice with established CD38-positive tumors. Mice deficient in T- and B-cells were injected subcutaneously with human tumor (Daudi) cells and once tumors reached an average volume of ∼200 mm3, mice were treated with 225Ac-daratumumab. To determine the localization of daratumumab within the tumor-bearing mice it was labeled with 111In, which can be easily imaged and is used as a surrogate to estimate the localization of 225Ac-daratumumab. The distribution of the 111In-daratumumab was then followed for 10 days using a microSPECT/CT scanner. To evaluate the antitumor ability of the 225Ac-daratumumab, tumor-bearing mice were injected with 225Ac- daratumumab at a dose of 400 nCi/0.3 μg of antibody, and 200 nCi/0.3 μg of antibody. As a control, mice were injected with either saline or an equivalent amount of unlabeled daratumumab. In addition, a group of mice was also treated with 30 times greater dose of unlabeled daratumumab (10 μg) - a dose which was previously shown to be effective against established tumors. 111Indaratumumab begun to accumulate in the tumor 24 hours after intraperitoneal injection and by 7 days was exclusively present in the tumor. The growth of the tumors in mice treated with 400 nCi/0.3 μg was significantly retarded compared to mice treated with equal concentration of unlabeled daratumumab or saline. Tumor growth was similar between mice treated with 400 nCi/0.3 μg of 225Ac-daratumumab and mice treated with 10 μg of unlabeled daratumumab. In conclusion, this study shows that labeling daratumumab with 225Ac increases its antitumor activity at least 30-fold. This study suggests that 225Ac labeling daratumumab increases its potency and could greatly reduce the amount of daratumumab needed for treatment in the clinic. This study also highlights the potential of targeting α-emitters to tumors as a viable therapeutic approach.