Burkitt's Lymphoma Raji Research Articles

Comparable efficacy of oral bendamustine versus intravenous administration in treating hematologic malignancies.

The purpose of this study was to analyze potential differences in antitumor efficacy and pharmacokinetics between intravenous (IV) bendamustine and a novel orally administered (PO) bendamustine agent that is utilizing the beneficial properties of superstaturated solid dispersions formulated in nanoparticles. Pharmacokinetics of IV versus PO bendamustine were determined by analysis of plasma samples collected from NSG mice treated with either IV or PO bendamustine. Plasma samples were analyzed using liquid chromatography-mass spectrometry following a liquid-liquid extraction to determine peak bendamustine concentration, area under the concentration-time curve, and the half-life in-vivo. In-vitro cytotoxicity of bendamustine against human non-Hodgkin Burkitt's Lymphoma (Raji), multiple myeloma (MM.1s), and B-cell acute lymphoblastic leukemia (RS4;11) cell lines was determined over time using MTS assays. Luciferase-tagged versions of the aforementioned cell lines were used to determine in-vivo bendamustine cytotoxicity of IV versus PO bendamustine at two different doses. Bendamustine at a high dose in-vitro causes cell death. There was no significant difference in antitumor activity between IV and novel PO bendamustine at a physiologically relevant concentration in all three xenograft models. In-vivo pharmacokinetics showed the oral bioavailability of bendamustine in mice to be 51.4%. The novel oral bendamustine agent tested exhibits good oral bioavailability and systemic exposure for in-vivo antitumor efficacy comparable to IV bendamustine. An oral bendamustine formulation offers exciting clinical potential as an additional method of administration for bendamustine and warrants further evaluation in clinical studies.

Effect of placental circulating T cells expressing CD16 on multiple hematological and solid tumor cancers through combination with various monoclonal antibodies.

2554 Background: PT-CD16VS is an allogeneic cell therapy derived from human postpartum placental circulating T (PT) cells that are genetically modified to express CD16 and endogenous T cell receptor (TCR) knockout. PT-CD16VS cells can be combined with various monoclonal antibodies to engage in anti-tumor antibody-dependent cellular cytotoxicity (ADCC) against diverse cancers with a “universal receptor” approach. Here we report the characterization and preclinical evaluation of PT-CD16VS in combination with monoclonal antibodies against both hematological and solid tumor cancers. Methods: PT cells were activated, transduced with a lentiviral vector containing a construct expressing a high affinity CD16 variant, CD16VS, and transfected to knock out the TCR. In vitro,functional activity of PT-CD16VS cells in combination with monoclonal antibodies was assessed in cytotoxicity, cytokine release, and proliferation assays. Invivo, PT-CD16VS was combined with Rituximab in a Raji xenograft model in NSG mice, and with Trastuzumab in a subcutaneous NCI-N87 xenograft model in NSG mice in which tumor volume was measured and tumor samples were evaluated for PT-CD16VS infiltration. Results: In vitro, PT-CD16VS exhibited potent ADCC and cytokine release when combined with Rituximab against CD20+ Burkitt's lymphoma (Daudi and Raji), with Trastuzumab against HER2+ gastric carcinoma (NCI-N87), with Trastuzumab or Avelumab against HER2+/PDL-1+ non-small cell lung carcinoma (NCI-H1975) and Trastuzumab-resistant metastatic mammary adenocarcinoma (JIMT-1), and with Cetuximab against EGFR+ triple negative metastatic mammary adenocarcinoma (MDA-MB-231). Moreover, examples of PT-CD16VS antigen-specific proliferation were demonstrated against Raji and NCI-N87 when cells were combined with Rituximab and Trastuzumab, respectively. In vivo, in combination with Rituximab in the Raji mouse model, PT-CD16VS exhibited significant survival benefit compared to vehicle and Rituximab alone treated groups. In the subcutaneous NCI-N87 solid tumor model, PT-CD16VS with Trastuzumab demonstrated significant reduction in tumor volume compared to vehicle, Trastuzumab, and Enhertu groups. In addition, PT-CD16VS infiltration into the tumor was shown to be Trastuzumab dependent, with infiltrating cells expressing high levels of CD16 and Ki67. Conclusions: Our results show that PT-CD16VS have potent in vitro and in vivo ADCC activity, and a single drug product has the potential to be combined with various monoclonal antibodies to target multiple cancers across hematological and solid tumor indications. This “universal receptor” with antibody-dependent targeting approach, together with the benefits of an allogeneic cell platform, may democratize accessibility of such therapies for patients.

Effect of Celastrus Orbiculatus Extract on proliferation and apoptosis of human Burkitt lymphoma cells.

The lymphoma incidence rate is on the rise, with invasive forms particularly prone to relapse following conventional treatment, posing a significant threat to human life and wellbeing. Numerous studies have shown that traditional Chinese botanical drug medicine offers promising therapeutic benefits for various malignancies, with previous experimental findings indicating that Celastrus orbiculatus extract effectively combats digestive tract tumors. However, its impact on lymphoma remains unexplored. This study aims to investigate the impact and underlying mechanisms of COE on the proliferation and apoptosis of Burkitt lymphoma cells. We diluted COE in RPMI-1640 medium to create various working concentrations and introduced it to human Burkitt lymphoma Raji and Ramos cells. To evaluate cell viability, we used the CCK-8 assay, and we observed morphological changes using HE staining. We also conducted Annexin V-PI and JC-1 staining experiments to assess apoptosis. By combining the cell cycle experiment with the EDU assay, we gained insights into the effects of COE on DNA replication in lymphoma cells. Using Western blotting, we detected alterations in apoptosis-related proteins. In vivo experiments revealed that following COE intervention, tumor volume decreased, survival time was prolonged, spleen size reduced, and the expression of tumor apoptosis-related proteins changed. Our findings indicate that COE effectively inhibits lymphoma cell proliferation and promotes apoptosis by regulating these apoptosis-related proteins.

Reversal Effect of NVP-BEZ235 on Doxorubicin-Resistance in Burkitt Lymphoma RAJI Cell Line

To study the reversal effect of NVP-BEZ235 on doxorubicin resistance in Burkitt lymphoma RAJI cell line. The doxorubicin-resistant cell line was induced by treating RAJI cells with a concentration gradient of doxorubicin. The levels of Pgp, p-AKT, and p-mTOR in cells were detected by Western blot. Cell viability was detected by MTT assay. IC50 was computed by SPSS. The doxorubicin-resistant Burkitt lymphoma cell line, RAJI/DOX, was established successfully. The expression of Pgp and the phosphorylation levels of AKT and mTOR in RAJI/DOX cell line were both higher than those in RAJI cell line. NVP-BEZ235 downregulated the phosphorylation levels of AKT and mTOR in RAJI/DOX cell line. NVP-BEZ235 inhibited the proliferation of RAJI/DOX cell line, and the effect was obvious when it was cooperated with doxorubicin. The constitutive activation of PI3K/AKT/mTOR pathway of RAJI/DOX cell line was more serious than RAJI cell line. NVP-BEZ235 reversed doxorubicin resistance of RAJI/DOX cell line by inhibiting the PI3K/AKT/mTOR signal pathway.

Abstract 1375: Abundance of KLRB1+ (CD161) T cells in anti-PD1 non responders coupled with enhanced tumor cytotoxicity of anti-CD161 (IMT-009) with anti-PD1 makes it a rational target for combination with anti-PD-(L)1 immunotherapy

Abstract Background: Intra-tumoral CD4+ and CD8+ T cells expressing inhibitory receptor CD161 have an effector memory phenotype with high cytotoxic potential. CD161 inhibits T and NK cell function in the presence of its ligand, CLEC2D. This suppression can be recovered by treatment with IMT-009, a first in class anti-CD161 antibody. Single cell transcriptomic analysis of anti-PD1 refractory microsatellite stable colorectal tumors identified presence of CD161+ CD4+ cytotoxic T cells and CD161+ PD1+ exhausted CD8 T cells suggesting that anti-CD161 treatment may provide benefit in these tumors both as a monotherapy and in combination with anti-PD1. It was also observed that CD161+ CD4/CD8T cells are more abundant in anti-PD1/L1 non responders compared with responders in hepatocellular carcinoma, cutaneous squamous cell carcinoma, clear cell renal carcinoma and breast cancer, providing a rationale for targeting CD161. Using engineered ex-vivo tumor models and dissociated human tumors, we have demonstrated that IMT-009 reverses CLEC2D mediated inhibition and restores primary T cell function. Additionally, co-treatment of IMT-009 with anti-PD1 further enhanced T cell mediated cytotoxicity, providing rationale for studies combining IMT-009 and anti PD1 in the clinic. Methods: Using the Snakemake pipeline developed in house, we processed publicly available scRNAseq data. Cells were clustered by Louvian method followed by cell type annotation by Seurat, and gene expression and gene signature levels per cluster were plotted. For ex-vivo assays, antigen specific CD8 T cells expanded from HLA-A2:01 donors were co-cultured with HLA-A2:01 expressing MDA-MB 231 triple negative breast cancer cell line or Raji Burkitt Lymphoma model with HLA-A2:01 allelic overexpression. Target tumor cells were engineered to express the ligands CLEC2D or PDL1. These cell lines were made to overexpress luciferase for monitoring target cell death using a luminescence readout. Additionally, dissociated tumor cells (DTCs) were cultured in presence of the treatment molecules. Intracellular staining of cytotoxic T cells along with detection of IFNγ, IL-2, TNFα and Granzyme B levels in culture supernatants by MSD/ELISA were used to assess activation profile of tumor infiltrated T cells. Results: Combining anti-PD1 and IMT-009 significantly enhanced cytotoxic benefit compared to individual monotherapy in both Raji and MDA-MB-231 target cell line models. IFNγ levels in supernatants of DTCs treated with this combination were elevated as compared to individual monotherapy. Together with scRNAseq data of increased abundance of KLRB1 (CD161) expressing T cells in anti PD1 non-responder patients, our data provide rationale for testing combination of IMT-009 with anti-PD1 in patients that may not respond to anti PD1 alone. Citation Format: Adwitiya Kar, Tim Nieuwenhuis, Meixue Duan, Siamak Tabrizi, Matthew Huggins, Elizabeth Scanlon, Cynthia Hession, Matthew Bernstein, Michael Ross, Ming Tang, Shruti Malu. Abundance of KLRB1+ (CD161) T cells in anti-PD1 non responders coupled with enhanced tumor cytotoxicity of anti-CD161 (IMT-009) with anti-PD1 makes it a rational target for combination with anti-PD-(L)1 immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1375.

Evaluation of potential role of R-loop and G-quadruplex DNA in the fragility of c-MYC during chromosomal translocation associated with Burkitt’s lymphoma

t(8;14) translocation is the hallmark of Burkitt's lymphoma and results in c-MYC deregulation. During the translocation, c-MYC gene on chromosome 8 gets juxtaposed to the Ig switch regions on chromosome 14. Although the promoter of c-MYC has been investigated for its mechanism of fragility, little is known about other c-MYC breakpoint regions. We have analyzed the translocation break points at the exon 1/intron 1 of c-MYC locus from patients with Burkitt's lymphoma. Results showed that the breakpoint region, when present on a plasmid, could fold into an R-loop confirmation in a transcription-dependent manner. Sodium bisulfite modification assay revealed significant single-strandedness on chromosomal DNA of Burkitt's lymphoma cell line, Raji, and normal lymphocytes, revealing distinct R-loops covering up to 100bp region. Besides, ChIP-DRIP analysis reveals that the R-loop antibody can bind to the breakpoint region. Further, we show the formation of stable parallel intramolecular G-quadruplex on non-template strand of the genome. Finally, incubation of purified AID invitro or overexpression of AID within the cells led to enhanced mutation frequency at the c-MYC breakpoint region. Interestingly, anti-γH2AX can bind to DSBs generated at the c-MYC breakpoint region within the cells. The formation of R-loop and G-quadruplex was found to be mutually exclusive. Therefore, our results suggest that AID can bind to the single-stranded region of the R-loop and G4 DNA, leading to the deamination of cytosines to uracil and induction of DNA breaks in one of the DNA strands, leading to double-strand break, which could culminate in t(8;14) chromosomal translocation.

The novel high-affinity humanized antibody IMM40H targets CD70, eliminates tumors via Fc-mediated effector functions, and interrupts CD70/CD27 signaling.

A significant level of CD70 can be detected in various types of tumor tissues and CD27 is expressed on Treg cells, but CD70 expression is low in normal tissues. The interaction between CD70 and CD27 can stimulate the proliferation and survival of cancer cells and increase the level of soluble CD27, which is associated with poor prognosis in patients with lymphoma and certain solid tumors. Thus, it is a promising therapeutic target for the treatment of many major CD70+ cancer indications, including CD70+ lymphoma, RCC, NSCLC, HNSCC and OC. IMM40H was obtained through hybridoma screening and antibody humanization techniques. IMM40H was evaluated for its binding, blocking, Fc-dependent effector functions and antitumor activity characteristics in various in vitro and in vivo systems. The safety and tolerability profile of IMM40H were evaluated through single and repeated administration in cynomolgus monkeys. In vitro cell-based assays demonstrated that IMM40H had considerably stronger CD70-binding affinity than competitor anti-CD70 antibodies, including cusatuzumab, which enabled it to block the interaction of between CD70 and CD27 more effectively. IMM40H also exhibited potent Fc-dependent effector functions (ADCC/CDC/ADCP), and could make a strong immune attack on tumor cells and enhance therapeutic efficacy. Preclinical findings showed that IMM40H had potent antitumor activity in multiple myeloma U266B1 xenograft model, and could eradicate subcutaneously established tumors at a low dose of 0.3 mg/kg. IMM40H (0.3 mg/kg) showed therapeutic effects faster than cusatuzumab (1 mg/kg). A strong synergistic effect between IMM01 (SIRPα-Fc fusion protein) and IMM40H was recorded in Burkitt's lymphoma Raji and renal carcinoma cell A498 tumor models. In cynomolgus monkeys, the highest non-severely toxic dose (HNSTD) for repeat-dose toxicity was up to 30 mg/kg, while the maximum tolerated dose (MTD) for single-dose toxicity was up to 100 mg/kg, confirming that IMM40H had a good safety and tolerability profile. IMM40H is a high-affinity humanized IgG1 specifically targeting the CD70 monoclonal antibody with enhanced Fc-dependent activities. IMM40H has a dual mechanism of action: inducing cytotoxicity against CD70+ tumor cells via various effector functions (ADCC, ADCP and CDC) and obstructs the proliferation and activation of Tregs by inhibiting CD70/CD27 signaling.

Regulatory Mechanism of Mangiferin Combined with Bortezomib on Malignant Biological Behavior of Burkitt Lymphoma and Its Effect on Expression of CXC Chemokine Receptors

To analyze the effects of mangiferin combined with bortezomib on the proliferation, invasion, apoptosis and autophagy of human Burkitt lymphoma Raji cells, as well as the expression of CXC chemokine receptors (CXCRs) family, and explore the molecular mechanism between them to provide scientific basis for basic research and clinical work of Burkitt lymphoma. Raji cells were intervened with different concentrations of mangiferin and bortezomib alone or in combination, then cell proliferation was detected by CCK-8 assay, cell invasion ability was detected by Transwell chamber method, cell apoptosis was detected by Annexin V/PI double-staining flow cytometry, apoptosis, autophagy and Akt/mTOR pathway protein expression were detected by Western blot, and the expression changes of CXCR family was detected by real-time quantitative PCR (RT-qPCR). Different concentrations of mangiferin intervened Raji cells for different time could inhibit cell viability in a concentration- and time-dependent manner (r =-0.682, r =-0.836). When Raji cells were intervened by combination of mangiferin and bortezomib, compared with single drug group, the proliferation and invasion abilities were significantly decreased, while the apoptosis level was significantly increased (P <0.01). Mangiferin combined with bortezomib could significantly up-regulate the expression of pro-apoptotic protein Bax and down-regulate the expression of anti-apoptotic protein Bcl-2 after intervention in Raji cells. Caspase-3 was also hydrolyzed and activated, and then induced the apoptosis of Raji cells. Mangiferin combined with bortezomib could up-regulate the expression of LC3Ⅱ protein in Raji cells, and the ratio of LC3Ⅱ/LC3Ⅰ in cells was significantly up-regulated compared with single drug or control group (P <0.01). Mangiferin combined with bortezomib could significantly inhibit the phosphorylation levels of Akt and mTOR, inhibit the proliferation and invasion of Raji cells by inhibiting Akt/mTOR pathway, and induce cell autophagy and apoptosis. Mangiferin and bortezomib could down-regulate the expressions of CXCR4 and CXCR7 mRNA after single-agent intervention in Raji cells, and the down-regulations of CXCR4 and CXCR7 mRNA expression were more significant when the two drugs were combined (P <0.01). Mangiferin alone or combined with bortezomib had no significant effect on CXCR5 mRNA expression in Raji cells (P >0.05), while the combination of the two drugs could down-regulate the expression of CXCR3 (P <0.05). Mangiferin combined with bortezomib can synergistically inhibit the proliferation and invasion of Raji cells, and induce autophagy and apoptosis. The mechanism may be related to the inhibition of Akt/mTOR signaling pathway, down-regulation of anti-apoptotic protein Bcl-2 and up-regulation of pro-apoptotic protein Bax, and the inhibition of the expression of CXCR family.

The Flavonoid Brusatol Induces Apoptosis in Aggressive Lymphoma Cells and Exhibits Synergistic Effect with Venetoclax

The most common lymphoid malignancies in adults are aggressive lymphomas, including Burkitt lymphoma and diffuse large B-cell lymphoma (DLBCL). Despite the available chemoimmunotherapy R-CHOP, one third of DLBCL patients still have primary refractory disease or relapse, and the incidence of these lymphomas continuously increases. Together, there is the great need for further development of new treatment strategies for aggressive lymphoma. One step forward seems to be Venetoclax (an FDA-approved Bcl-2 inhibitor), which showed an overall response rate of 44% in a phase 1 trial. Here, we evaluated a flavonoid - Brusatol, which showed anti-tumour activity in various preclinical cancer models, to improve anti-lymphoma therapies. We investigated the potential of Brusatol in the treatment of aggressive lymphoma cells as a single agent or in combination with other drugs, including Venetoclax. Seven cell lines representing different types of lymphoma (BL2, Raji (Burkitt lymphoma), SuDHL4, Karpas422 (GCB-DLBCL), RI-1, U2932 (ABC-DLBCL) and Jurkat (T-cell leukemia)) were treated with increasing concentrations of Brusatol (up to 10µM) for 72h to determine the IC50 values. Based on these results, 50nM and 250nM Brusatol were selected for further experiments. Cells were treated with Brusatol for 24h and 48h and apoptosis induction (Annexin V staining, Caspase-3- and PARP-cleavage) and changes in cell cycle (PI staining) were assessed. Moreover, samples from untreated and Brusatol-treated cells were collected for Western blot analysis. To investigate potential effects of Brusatol on protein synthesis in lymphoma cells, four cell lines (BL2, SuDHL4, RI-1 and U2932) were treated for 4h, and then the nascent protein synthesis was evaluated by detection of an incorporated methionine analogue using click-chemistry. Furthermore, co-treatment of Brusatol with inhibitors of Bcl-2 (Venetoclax), Bcl-XL (A-1331852), Mcl-1 (S63845), BTK (Ibrutinib), PI3K (Idealisib), Myc (EN4, JQ1), respectively, was performed in the same four cell lines and Annexin V levels were measured after 24h. Finally, the effect of combining Brusatol with Venetoclax was determined using the Bliss independence model for SuDHL4 and U2932 after 24h treatment via TMRE staining. In all investigated cell lines, Brusatol caused cell growth inhibition in a concentration-dependent manner. However, based on the results of the apoptosis assays, they could be grouped into cell lines more (BL2, Jurkat, Raji, SuDHL4) and less (Karpas422, RI-1, U2932) sensitive to Brusatol. Moreover, cell cycle analysis revealed an increase of cells in the sub-G1 phase in the more sensitive cell lines, indicating the induction of cell death. Western blot analysis of more sensitive cell lines showed decreased levels of the prosurvival proteins Bcl-2, Bcl-XL and Mcl-1. Additionally, we detected reduced p53 and Myc protein expression in cell lines more sensitive to Brusatol after 24h treatment. Notably, the protein expression profile of untreated cells indicates that cell lines with higher Myc levels are more sensitive to Brusatol treatment. Furthermore, mRNA expression analysis revealed that the reduction of affected proteins mainly occurred on protein level. Therefore, we investigated the effect of Brusatol on protein biosynthesis and observed protein translation inhibition in all tested cell lines. Interestingly, increased induction of apoptosis was apparent in all cell lines upon co-treatment with Brusatol and Bcl-XL inhibitor, in three of the four cell lines treated with the combination of Brusatol and Bcl-2 inhibitor, and in two cell lines when Brusatol was combined with Mcl-1 inhibitor. In contrast, combining Brusatol had no beneficial effect in combination with all other drugs tested. Finally, analysis of the Bliss independent model showed that the combination of Brusatol and Venetoclax synergistically enhanced the killing of lymphoma cells. Our data indicate that Brusatol efficiently induces cell death in aggressive lymphoma cells by reducing the expression of prosurvival proteins. Interestingly, cells with higher Myc levels were especially sensitive to Brusatol. Additionally, the combination of Brusatol with Venetoclax results in enhanced induction of apoptosis. Thus, our study suggests that Brusatol, alone or in combination with Venetoclax, represents a very interesting agent for further development of novel anti-lymphoma therapies.

Pre-clinical efficacy of CD20-targeted chimeric antigen receptor T cells for non-Hodgkin's lymphoma

BackgroundA 4-1BB/CD3-ζ-costimulated CAR-T against CD20 (CAR-T20) was subjected to a systemic efficacy evaluation in a cell co-culture model, and NOD-SCID IL-2 receptor gamma null mice (short for NSG mice) were xenografted with human Burkitt's lymphoma Raji cells.MethodsCAR-T20 cells were incubated with target cells (K562, K562 CD20 or Raji cells) at ratios of 10:1 and 5:1 for 24 h, and the killing rate was estimated by an LDH cytotoxicity assay. To evaluate the effect of CAR-T20 on the survival time of tumor-bearing animals, 30 NSG mice were employed, and Raji-Luc cells (5 × 105 cells per mouse) were administered prior to CAR-T20 administration. The survival time, optical intensity of Raji-Luc cells, clinical symptoms, and body mass of the animals were observed. Another 144 male NSG mice were employed to investigate the proliferation and antitumor effects of CAR-T20. Human cytokine and murine cytokines were detected at 1, 7, 14, 21, 28, 42, 56 and 90 days post-CAR-T administration, while biochemistry index analysis, T-cell and CAR-T-cell detection in peripheral blood, and histopathological examination were performed at 14, 28, 56 and 90 days post-administration.ResultsCAR-T20 cells had a specific killing effect on CD20-expressing cells in vitro. At a dose of 1 × 106 per mouse or above, CAR-T20 prolonged the median survival time from 14 days to more than 3 months, inhibited the proliferation of Raji cells in mice, and alleviated the clinical manifestations and weight loss caused by the Raji-Luc cell load. CAR-T20 at a dose of 2 × 106 per mouse or above inhibited the proliferation of Raji cells in mice for up to 111 days post-administration without recurrence. The numbers of T cells and CAR-T cells in the animals administered CAR-T20 increased significantly when Raji cells were markedly proliferated and subsequently decreased when Raji cells were predominantly inhibited. CAR-T20 increased human IFN-γ, murine TNF and murine IL-6 levels and decreased human IL-10 levels in tumor-bearing mice. The incidences of xenografted tumors in organs/tissues were also reduced effectively by CAR-T20.ConclusionThe effective dose of CAR-T20 in mice starts from 1 × 106 per mouse, equivalent to a clinical dose of 5 × 106/kg. Together, our data support the clinical translation of CAR-T20 for R/R B-cell NHL patients.

Bioymifi suppresses Burkitt’s lymphoma cell line proliferation via cell cycle arrest and apoptosis

Purpose: To find a new treatment alternative for Burkitt’s lymphoma, one of the B cell-derived non-Hodgkin's lymphomas that has an extremely aggressive growth profile with a high mortality rate.&#x0D; Methods: Bioymifi (death receptor agonist) was administered at various doses to Human Burkitt's Lymphoma Raji cell lines, and cell proliferation was evaluated using XTT analysis. Apoptosis induction and cell cycle analysis were assessed using flow cytometry.&#x0D; Results: Bioymifi exhibited good cytotoxicity in Raji cell lines with IC50 value of 29.5 μM. Furthermore, results of apoptosis induction and cell cycle analysis indicated that Bioymifi had potent anti-proliferative activity through sub-S cell cycle arrest and stimulation of apoptosis (p &lt; 0.05).&#x0D; Conclusion: Bioymifi has anticancer potentials against Human Burkitt's Lymphoma Raji cell lines. Further pharmacological studies will be required to establish the mechanism of action.

Formation of Lymphoma Hybrid Spheroids and Drug Testing in Real Time with the Use of Fluorescence Optical Tweezers.

Interactions between stromal and lymphoma cells in the bone marrow are closely related to drug resistance and therapy failure. Physiologically relevant pre-clinical three-dimensional (3D) models recapitulating lymphoma microenvironmental complexity do not currently exist. In this study, we proposed a scheme for optically controlled hybrid lymphoma spheroid formation with the use of optical tweezers (OT). Following the preparation of stromal spheroids using agarose hydrogel, two aggressive non-Hodgkin lymphoma B-cell lines, Ri-1 (DLBCL) and Raji (Burkitt lymphoma), were used to conduct multi-cellular spheroid formation driven by in-house-developed fluorescence optical tweezers. Importantly, the newly formed hybrid spheroid preserved the 3D architecture for the next 24 h. Our model was successfully used for the evaluation of the influence of the anticancer agents doxorubicin (DOX), ibrutinib (IBR), and AMD3100 (plerixafor) on the adhesive properties of lymphoma cells. Importantly, our study revealed that a co-treatment of DOX and IBR with AMD3100 affects the adhesion of B-NHL lymphoma cells.

Bryostatin Activates CAR T-Cell Antigen-Non-Specific Killing (CTAK), and CAR-T NK-Like Killing for Pre-B ALL, While Blocking Cytolysis of a Burkitt Lymphoma Cell Line.

The advent of CAR-T cell therapy has changed the face of clinical care for relapsed and refractory pre-B-acute lymphocytic leukemia (B-ALL) and lymphoma. Although curative responses are reported, long-term cures remain below 50%. Different CAR T-cell leukemia targets appear to have different mechanisms of CAR-T escape. For CD22, therapeutic evasion is linked to down-modulation of the number CD22 proteins expressed on the extracellular aspect of the leukemia cell plasma membrane. Recently, pharmacologic agents known to induce cellular differentiation or epigenetic modification of leukemia have been shown to impact CD22 and CD19 expression levels on B-ALL, and thereby increase sensitivity to CAR-T mediated cytolysis. We explored the impact of epigenetic modifiers and differentiation agents on leukemia cell lines of B cell origin, as well as normal B cells. We confirmed the activity of bryostatin to increase CD22 expression on model cell lines. However, bryostatin does not change CD22 levels on normal B cells. Furthermore, bryostatin inhibited CAR-T mediated cytolysis of the Raji Burkitt lymphoma cell line. Bryostatin increased the cytolysis by CD22 CAR-T for B-ALL cell lines by at least three mechanisms: 1) the previously reported increase in CD22 target cell numbers on the cell surface, 2) the induction of NK ligands, and 3) the induction of ligands that sensitize leukemia cells to activated T cell antigen-non-specific killing. The opposite effect was seen for Burkitt lymphoma, which arises from a more mature B cell lineage. These findings should caution investigators against a universal application of agents shown to increase killing of leukemia target cells by CAR-T in a specific disease class, and highlights that activation of non-CAR-mediated killing by activated T cells may play a significant role in the control of disease. We have termed the killing of leukemia targets, by a set of cell-surface receptors that does not overlap with NK-like killing “CTAK,” CAR-T Cell antigen-non-specific killing.

A Novel Method for Highly Efficient Ex Vivo Expansion and Genetic Engineering of Human Natural Killer Cells for Cancer Therapy

Cancer immunotherapy with adoptive transfer of human leukocyte antigen-mismatched, CD19-targetd chimeric antigen receptor (CAR)-transduced natural killer (NK) cells has attracted attention because of its efficacy and safety when infused in patients with refractory and relapsed B-cell lymphomas. However, generating clinical doses of CAR-NK cells is still a challenge. The methods for ex vivo expansion and genetic modification of primary human NK cells usually rely on the use of irradiated feeder cell lines, which has been restrictive due to high costs, scale-up difficulties, and licensing restrictions. Hence, novel strategies that do not require feeder cells will be beneficial in standardizing these types of cell therapies. In this study, we show the effectiveness of a novel feeder-free culture system in expanding NK cells ex vivo and generating CAR-NK cells.Unsorted peripheral blood mononuclear cells (PBMCs) collected from healthy donors were cultured with a reagent containing dissolvable microspheres that are conjugated with anti-CD2/NKp46 antibodies (Cloudz TM Human NK Cell Expansion Kit) and a combination of multiple cytokines, including interleukin (IL)-2, IL-12, IL-18, and IL-21 in medium supplemented with 10% fetal bovine serum. The activated NK cells were transduced using the RD114-pseudotyped retrovirus vector.To test whether the reagent promoted ex vivo NK cell expansion, we cultured PBMCs from 13 donors. The percentage of NK (CD56 + CD3 -) cells of initially isolated PBMCs was 15.3±7.5%. In the presence of multiple cytokine combinations, NK cell purity gradually increased and reached 91.6±7.6% by day 21. The NK cells expanded to 75.6±59.2-fold at day 10, 334±217-fold at day 14 and 1,542±913-fold at day 21. The expanded NK cells degranulated and produced intracellular cytokines upon exposure to K562 myeloid leukemia cells. The NK cells efficiently killed myeloid leukemia cells, such as K562, THP1, and KG1. The expression pattern of killer cell immunoglobulin-like receptors on NK cells remained unchanged. The expression of activating NK cell receptors, including NKp30 and NKp44, increased after 21 days of culture.Thereafter, a gene transfer to the primary human NK cells was conducted. We tested transduction efficiency and yields of modified cells on 7 days after the procedure by empty-vector transduction into NK cells expanded ex vivo for 3, 6, and 10 days (n=3, each). The results were presented as means ± standard deviation; 55.6±11.6%, 61.6±14.1%, and 73.6%±6.2% for GFP positivity in NK cells and 5.7±1.5 folds, 56.4±42.2 folds, and 12.7±5.8 folds for yields of modified cells. We selected the condition in which the transduction was carried out using NK cells expanded ex vivo for 6 days, although the differences were not statistically significant. Next, anti-CD19 CAR with a 4-1BB costimulatory and CD3z domain was transduced into NK cells. We confirmed high transduction efficiency (59.8% ±20.5%, n=3) and high CAR protein expression on the cell surface, while NK cells maintained their purity and minimal T cell outgrowth was observed. CAR-NK cells maintained their proliferative status and further expanded 15.2±6.4-fold after 7 days of the procedure.To determine whether the generated anti-CD19 CAR-NK cells had a specific effect on B cell malignancies, a CD107a mobilization assay, intracellular cytokine assay, and a flow cytometry-based cytotoxicity assay was employed. We found that CAR transduction could render NK cells to generate specific and powerful responses against CD19-positive, NK-resistant leukemia and lymphoma cell lines, such as BCR-ABL-positive acute lymphoblastic leukemia (ALL) OP-1, Burkitt lymphoma Raji, and KMT2A-rearranged ALL RS4;11, at various effector: target (ET) ratios. For example, in a 4 hour -assay, the cytotoxic effects of anti-CD19 CAR-NK cells showed 86.9±0.2% cytotoxicity against OP-1, while mock NK cells showed 17.4±2.9% cytotoxicity (ET ratio 1:1).In conclusion, this study revealed highly efficient functions of the novel feeder-free culture system, including highly efficient ex vivo expansion of primary human NK cells and generation of genetically modified NK cells for cancer immunotherapy. In future studies, we will investigate large-scale cultures using specialized flasks and GMP-grade reagents for clinical translation and the in vivo activities of the cell products in mouse xenograft models. DisclosuresImai: Juno Therapeutics: Patents & Royalties: chimeric receptor with 4-1BB signaling domain.

Targeting Burkitt's Lymphoma Using Novel, Discrete Carbon Nanotubes

Burkitt's lymphoma is an incredibly aggressive B-cell derived non-Hodgkin lymphoma that is quickly fatal if left untreated. Doxorubicin (Doxo) provides one method of treatment for this cancer, but has numerous common side effects including significant weight loss, nausea, hair loss, bone marrow suppression, vomiting, rash, heart damage, allergic reaction, tissue damage at injection site, and treatment-related leukemia. Therefore, better therapies which deliver Doxo specifically to tumor cells hold great promise for a more effective therapy with reduced side-effect severity. Carbon nanotubes (CNTs) have long been of interest as drug delivery transporters because of their unique structure, high drug-binding surface area, and variety of customization options. Unfortunately, most CNTs have proven toxic during in vivo studies because of the presence of high residual contaminants and near-unavoidable aggregative properties. A novel form of clean, discrete CNT (dCNT) marketed as Medical Grade MOLECULAR REBAR® (MGMR®) lack the aggregative problems and high toxin levels of CNT before them and show promise as a drug delivery technology.We observed no toxicity of dCNTs in vitro across multiple cell types or in vivo in mice establishing a high MTD of at least 70 mg/kg by an i.v. route. Biodistribution studies of Lu-177 radioisotope labeled dCNTs (single tail vein injection into BALB/C female mice) demonstrated tolerability and clearance from various organs. Mice were sacrificed at 7 timepoints under 34d and many organs were analyzed for accumulation. The liver, spleen, and blood identified one route of clearance and distribution, but it was interesting to note that the bone retained ~10% of injected dCNTs at all time points, demonstrating potential for bone targeted therapies. Further targeting to the bone was possible by grafting commonly available bone chelators to the surface of the dCNT to create BT-dCNT as shown by significantly great adhesion (p<0.05) to ex vivo bone.Protocols were then developed to saturate the surface of the dCNT with therapeutic Active Pharmaceutical Ingredients (API) and then characterize their elution/release over time. We then optimized methods for drug-loading and observed a slow drug release profile for most drugs. Enhanced pharmacokinetic information was tested in cell culture and then confirmed by animal experimentation. In addition, cell penetration and delivery study of MGMR® in vitro showed that endocytosis is nontoxic and well tolerated and that small peptides can be delivered to cancer cells and retain their functionality. Zeta Potential measurements (-40 to -50 mV) and Dynamic Light Scattering validated the stability of different dCNTs over time.Lastly, in a Raji-Burkitt's lymphoma mouse model, we saw significant increases in survival, and decreases in tumor burden in mice treated with Doxo (free, in dCNTs, and in BT-dCNTs vs no drug control). SCID mice were inoculated via the tail vein with 2x105 Raji B. luc+ tumor cells and treated once a week with one of the following: Doxo (5 mg/kg), dCNTs (14.29 mg/kg), dCNTs (14.29 mg/kg) + Doxo (5 mg/kg), BT-dCNTs (14.29 mg/kg) + Doxo (5 mg/kg), or no treatment. Using whole body bioluminescence imaging (BLI), we saw that the groups receiving both the Doxo-loaded dCNTs and the Doxo-loaded BT-dCNTs had greatly reduced tumor burden over time compared to untreated groups, and a tumor burden similar to those that received free Doxo alone (Figure 1A). Both groups receiving Doxo-loaded dCNTs also showed increased survival compared to the Doxo alone group (Figure 1B). Importantly, the typical signs of Doxo toxicity in mice, characterized by rapid weight loss or drug-related mortality, were not seen in the groups that received Doxo-loaded CNTs (Figure 1C).In summary, dCNTs is a highly promising biomedical tool for drug delivery for clinical applications. Our dCNTs represent a promising platform for use in biomedical research as they have proven to be nontoxic, tunable, stable, and capable of drug delivery and organ-specific targeting. This novel technology gives great promise for the bone-targeting ability of novel carbon nanotubes in vivo as a bone-specific drug delivery mechanism for diseases that grow in bone or bone marrow specifically. [Display omitted] DisclosuresFalank:BioPact Ventures, LLC: Research Funding. Tassett:BioPact Ventures, LLC: Employment. Harris:BioPact Ventures, LLC: Research Funding. Farrell:BioPact Ventures, LLC: Research Funding. Everill:BioPact Ventures, LLC: Employment. Reagan:BioPact Ventures, LLC: Research Funding.

CAR-T Cells Targeting BAFF-Receptor for B-Cell Malignancies: A Potential Alternative to CD19

Background: Chimeric antigen receptor (CAR) T-cell adoptive therapies have the potential to revolutionize the treatment of cancers. Particularly in hematological malignancies, there are reports of promising clinical outcomes in advanced non-Hodgkin lymphomas (NHLs) with CD19-CAR T-cell therapy. However, disease relapse is problematic, and is thought to be caused by poor long-term persistence of the CAR T-cells, and loss of the CD19 target on tumors. Thus, there is an urgent need for improved novel CAR T-cell therapies directed at alternative targets.The CAR T-cell platform relies on antibody-derived single chain fragments (scFv), which are genetically engineered into chimeric T-cell receptors, and that recognize target cell surface proteins on tumors. One potential target is B-cell activating factor receptor (BAFF-R), a tumor necrosis factor receptor superfamily protein (TNFRSF13C) specifically involved in B lymphocyte development and mature B-cell survival, that is primarily expressed on B cells and various subtypes of B-cell NHLs and ALL. We have recently developed a humanized therapeutic BAFF-R antibody with strong anti B-cell tumor activity.Method and Results: We adapted a scFv based on our humanized anti-BAFF-R antibody onto a second generation CAR platform containing CD3ζ and 4-1BB intracellular signaling domains. In response to BAFF-R-expressing malignant human B cells (NHLs, acute lymphoblastic leukemias, and chronic lymphocytic leukemias), our CAR T cells readily proliferated and secreted cytotoxic cytokines. We demonstrated both significant levels of BAFF-R CAR T-cell activation and malignant B-cell killing in vitro .Established human NHL tumors in xenogeneic models were eliminated following BAFF-R CAR T-cell treatments in vivo . Remarkable tumor-free survival was repeatedly observed in human lymphoma xenograft models including JeKo-1 (mantle cell lymphoma) and Raji (Burkitt lymphoma) in NSG mice. Moreover, upon tumor re-challenge and without any further treatment tumors failed to develop, due to persisting CAR T cells conferring continued anti-lymphoma activity. We pursued optimization of CAR T-cell persistence by comparing three subsets of early stage T cells (central memory, TCM; memory stem, TSCM; and naïve, TN) as potential starting material for CAR T cell generation. Our in vivo studies show CAR T cells from the TN population retained highest potency eradicating established tumors at a minimal therapeutic dose compared to other subtypes; 80% of TN CAR treated mice achieved long-term survival compared to 20% of TCM and 40% of TSCM CAR treated mice. We also observed the long term anti-tumor effects conferred by CD8+ CAR T cells required the addition of CD4+ CAR T cells. Finally, we performed a head-to-head comparison of BAFF-R CAR T cells with CD19 CAR T cells in the Raji model (Figure). Comparable expression levels of BAFF-R and CD19 were observed on the tumor cells (A), and both treatments elicited significant antitumor effects compared to T-cell and saline controls (B-C). However, the BAFF-R CAR T-cell treatment demonstrated long-term tumor free survival in all treated mice compared to the CD19 CAR T-cell treated cohort, which only showed delayed tumor growth (P<0.01, B-C).Conclusion: BAFF-R CAR T cells demonstrate remarkable efficacy against B-cell malignancies. Unexpectedly, we found that naïve T cells were superior to TCM or TSCM subpopulations as starting material in vivo . Naïve T-cell starting products, after transduction and expansion, generated sufficient numbers of TCM and TSCM CAR T cells for activity. Targeting BAFF-R combined with our strategy to improve CAR T-cell persistence potentially addresses unmet clinical needs in B-cell NHLs and ALL, particularly in the setting of CD19 CAR-T-resistance or CD19-negative relapse. [Display omitted] DisclosuresNeelapu:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Karus: Research Funding; Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectis Inc.: Research Funding; Poseida Therapeutics, Inc: Research Funding. Kwak:InnoLifes: Consultancy, Equity Ownership; Pepromene Bio: Consultancy, Equity Ownership; Celltrion, Inc: Consultancy.

Mechanism of Pax5 Down-Regulation and Its Function in Primary Effusion Lymphoma

Introduction: Primary effusion lymphoma (PEL) is a subtype of aggressive non-Hodgkin lymphoma caused by Kaposi sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 (HHV-8). PEL develops body cavity effusion without tumor masses especially in immunodeficient patients. The prognosis of PEL is very poor ad there is no standard therapy for PEL. Therefore, the molecular pathogenesis of PEL is needed to be clarified to develop novel therapy. The expressions of Pax5 in PEL patient samples were decreased compared with KSHV/HHV-8-negative B-cell lines in the publically available data set. In this study, we analyzed the mechanism of Pax5 down-regulation and performed the restoration of Pax5 in PEL cells.Methods:Pax5 mRNA expressions of eight PEL cell lines were examined by polymerase chain reaction (PCR). To study the function of Pax5 in PEL, Pax5 was conditionally expressed using tet-on system in three PEL cell lines. To examine the mechanism of Pax5 reduction in PEL, the bisulfite sequencing was performed to analyze the methylation of the Pax5 promoter region in PEL cells. The DNA methyltransferase inhibitor, 5-Azacytidine, was used for demethylation of Pax5 promoter. The anti-proliferative activities of restoring Pax5 in PEL cell lines were analyzed by the methylthiotetrazole method. Cell cycle profiles were analyzed using flow cytometry. Microarray analysis was performed to detect Pax5-regulated genes. The master regulator of KSHV/HHV-8 viral replication, ORF50 , was determined by quantitative PCR.Results: The expression of Pax5 mRNA in PEL cell lines was weaker than in the Burkitt lymphoma cell line, Raji, and normal B-cells derived from peripheral blood mononuclear cells, which express Pax5 mRNA. Although Pax5 promoter region of Raji cells was not methylated, that of PEL cells was heavily methylated. The treatment with 5-Azacytidine induced the demethylation of Pax5 promoter region and restored the expression of Pax5 mRNA. Restoration of Pax5 suppressed cell proliferation via cell cycle arrest. Cell cycle-related genes were down-regulated and several B-cell markers were up-regulated in Pax5-induced PEL cells. Pax5 did not induce ORF50 expression.Conclusions:Pax5 is silenced in PEL cells via the methylation of Pax5 promoter region. Pax5 regulates cell cycle and causes growth inhibition. Reduction of Pax5 has a critical role in the development of PEL and Pax5 is a tumor suppressor in PEL. The targeting of Pax5 with DNA methyltransferase inhibitor could be a novel therapeutic approach in patients with PEL. DisclosuresNo relevant conflicts of interest to declare.

Dual Inhibition of Aurora Kinase and BRD4 Leads to Improved Anti-Tumor Activity in Myc-Overexpressing Lymphoma Cells

Background:Myc has proven extremely difficult to target therapeutically. Therefore, we hypothesized that optimal inhibition of multiple key pathways involved in Myc expression could overcome this long-standing problem. We identified aurora A kinase (AURKA) and bromodomain protein (BRD)4 as therapeutic targets to counteract Myc deregulation based on strong evidence that these pathways are essential for tumor maintenance in Myc-driven malignancies.Methods: Cytotoxicity assays using MTS and trypan blue were used to compare levels of drug sensitivity in lymphoma cell lines with high and low Myc mRNA expression. Apoptosis and cell cycle assays were performed using Annexin V and Propidium Iodide staining. Cycloheximide chase followed by immunoblotting was used to examine Myc protein degradation kinetics, and real-time RT-PCR was used to examine Myc mRNA levels. Murine xenograft models were used to assess the efficacy and tolerability of single vs. combined inhibition.Results: Myc-overexpressing Burkitt lymphoma (Raji) cells were treated with increasing concentrations of small molecule inhibitors against AURKA (MLN-8237), BRD4 (I-BET-151), or both drugs for 48 to 72 hours and cell viability was evaluated. MLN-8237 and I-BET-151 inhibited cell growth individually with IC-50 of 10 nM, and 650 nM, respectively. As compared to treatment with the single agent alone, dual treatment induced more significant cell growth inhibition with IC-50 achieved at drug concentrations of < 5 nM and 500 nM for MLN-8237 and I-BET-151, respectively. The combination index (CI) values were less than 1 at various drug concentrations, indicating that different combinations were synergistic in terms of inhibitory effect on tumor cell viability. Superior activity of the dual inhibition was also noted in different Myc-overexpressing lymphoma cells. Combination treatments increased apoptosis and induced more pronounced cell cycle arrest compared to the single agent treatment alone.We then analyzed protein expression by Western blot. MLN-8237 reduced the expression of p-HisH3 and p-AURKA while increasing the expression of p-S6K. Treatment with I-BET-151 resulted in significant reduction of Myc total protein. Cycloheximide chase experiments demonstrated that inhibition of AURKA increased the rate of Myc protein degradation, while BRD4 inhibition had no effect on degradation kinetics. Conversely MLN-8237 did not decrease Myc transcription, while I-BET-151 significantly reduced the levels of Myc mRNA. The combination of the two drugs demonstrated additive/synergistic decreases of both Myc protein degradation and Myc transcription at higher drug doses.Lastly, athymic nude mice bearing Ramos lymphoma xenografts were treated with MLN-8237, I-BET-151, or with the combination of both agents (n = 8 per group). The mean tumor volumes in mice treated with negative control, I-BET-151, and MLN-8237 as single agents were 3480, 2320, and 671 mm3, respectively, at Day 28. Mice treated with MLN-8237/I-BET-151 combinations had mean tumor volume of 461 mm3 at Day 28. The survival rates for mice treated with negative control, I-BET-151, and MLN-8237 as single agents were 0%, 10%, and 70%, respectively, at Day 35 (Figure 1). Mice treated with the MLN-8237 and I-BET-151 combination demonstrated a 100% survival rate at Day 35.Conclusion: Our data demonstrated that Myc-overexpressing lymphoma can be successfully targeted by inhibition of Myc transcription and/or enhancement of Myc protein degradation. Specifically, MLN-8237, a small molecule inhibitor against AURKA, induced apoptosis of Myc-overexpressing tumor cells in vitro and showed the most promising anti-tumor activity in mice bearing Myc-overexpressing lymphoma, especially when combined with I-BET-151, a BRD4 inhibitor. [Display omitted] DisclosuresPark:Cornerstone Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gilead: Speakers Bureau; Seattle Genetics: Research Funding; Teva: Consultancy, Research Funding; Takeda: Research Funding. Ghosh:TG Therapeutics: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding, Speakers Bureau; Jassen: Consultancy, Honoraria, Research Funding; Kite Pharma: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Speakers Bureau; Seattle Genetics: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Speakers Bureau; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau. Sharpless:Unity Biotechnology: Other: NA; HealthSpan Diagnostics: Other: NA; Pfizer: Other: NA; G1 Therapeutics: Equity Ownership.

Construction and Characterization of 1F5 Chimeric Anti-CD20 Monoclonal Antibodies: an Efficient Splicing by Overlap Extension-polymerase Chain Reaction Technique

Despite the unparalleled success of anti-CD20-targeted immunotherapy, the currently available mAbs are not sufficiently efficacious in the treatment of lymphoma. 1F5 is one of a panel of anti-CD20 mAbs that was used in the B-cell lymphoma serotherapy. Despite the efficacy of murine 1F5 mAbs in lymphoma patients, the 1F5 chimeric antibodies with human effector functionality are yet to be approved and widely used in the treatment of lymphoma. In this study, the conversion of 1F5 mAb from mouse IgG2a to human-mouse chimeric IgG1 was achieved and the chimeric antibody was partially characterized. We constructed the 1F5 chimeric mouse-human anti-CD20 antibody genes using an efficient Splicing by overlap extension-polymerase chain reaction (SOE-PCR) technique and cloned the chimeric heavy and light genes in pBudCE4.1 mammalian expression vector, followed by purification of the expressed chimeric 1F5 mAbs using affinity chromatography. Our investigation also included the biological properties of purified chimeric antibodies. The generated 1F5 chimeric mAbs mediate complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) against Raji and Daudi Burkitt's lymphoma cell lines, which were comparable with rituximab and exhibit superior reduction in cell viability in vitro, compared to rituximab. The current study indicated that the generated chimeric 1F5 mAbs has potential CDC and ADCC activity which was comparable with rituximab whereas it exhibits a superior reduction in cell viability, compared to rituximab. Our work contributes to future studies involving in vivo biological functions and the application of the 1F5 chimeric antibody.

Selenium nanoparticles: Synthesis, in-vitro cytotoxicity, antioxidant activity and interaction studies with ct-DNA and HSA, HHb and Cyt c serum proteins

The aim of this study was the synthesis of selenium nanoparticles (SeNPs) employing vitamin C as a biocompatible and low toxic reducing agent. The synthesized selenium nanoparticles were characterized by using UV–vis, FT-IR, SEM-EDX, TEM, DLS, and zeta potential measurements. The results of the DPPH free radical scavenging assay demonstrate that this synthesized nano-selenium has strong potentials to scavenge the free radicals and cytotoxicity against MCF-7 and Raji Burkitt's lymphoma cancer cell lines. The interaction of calf thymus DNA (ct-DNA) with SeNPs indicated that the anticancer activity might be associated with the DNA-binding properties of nano-selenium. Finally, it was found that the synthesized nano-selenium can bind to the most important blood proteins such as human serum albumin (HSA), human hemoglobin (HHb), and Cytochrome c (Cyt c). The results showed that the secondary structure of these proteins remains unchanged, suggesting that the synthesized nano-selenium could be employed as a carrier in the drug delivery system without any cytotoxicity effect.