Lymphoma Cell Line Raji Research Articles

A new compound, phomaherbarine A, induces cytolytic reactivation in epstein-barr virus-positive B cell lines

Epstein-Barr virus (EBV), the first virus found to induce cancer in humans, has been frequently detected in various types of B cell lymphomas. During its latent phase, EBV expresses a limited set of proteins crucial for its persistence. Induction of the lytic phase of EBV has shown promise in the treatment of EBV-associated malignancies. The present study assessed the ability of phomaherbarine A, a novel compound derived from the endophytic fungus Phoma herbarum DBE-M1, to stimulate lytic replication of EBV in B95-8 cells. Phomaherbarine A was found to efficiently initiate the expression of both early and late EBV lytic genes in B95-8 cells, with this initiation being further heightened by the addition of phorbol myristate acetate and sodium butyrate. Moreover, phomaherbarine A demonstrated notable cytotoxicity against the EBV-associated B cell lymphoma cell lines B95-8 and Raji. Mechanistically, phomaherbarine A induces apoptosis in these cells through the activation of caspase-3/7. When combined with ganciclovir, phomaherbarine A does not interfere with the reduction of viral replication by ganciclovir and sustains its apoptosis induction. In conclusion, these findings indicate that phomaherbarine A may be a promising candidate for therapeutic intervention in patients with EBV-associated B cell lymphomas.

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 2730: Beyond antibodies and CAR-T: Topologically-engineered, super-dimeric antibody-like molecules with dual Fc domains for trispecific, bivalent targeting of CD19, CD20, and Fcgamma receptors

Abstract Background: Depletion of B cells has resulted in major therapeutic benefits in autoimmune diseases and hematological malignancies. Several FDA-approved B cell-depleting antibody-based therapies are available, including antibody-drug conjugates, T cell engagers, CAR-T cells, and antibody variants with enhanced effector functions through Fc engineering. However, a significant unmet need remains in both oncology and autoimmune disease. All currently approved B cell-depleting therapies target a single B cell antigen, increasing potential for incomplete depletion and emergence of escape mutants that give rise to relapse and treatment failure. Methods: Here we describe trispecific antibody-like molecules generated using GEM-DIMER technology. In addition to bivalent target binding of both CD19 and CD20, the GEM-DIMER candidates are designed to have two Fc domains to enable powerful effector functions via cooperative binding of Fcgamma receptors. The approach is based on incorporation of a super-dimerization domain into the hinge region of an immunoglobulin heavy chain, enabling the combination of all of the components of two IgG antibodies into a single molecule. Using this approach, we generated GEM-DIMER candidates from rituximab and FMC63, the parental anti-CD19 antibody for anti-CD19 scFv used in approved CAR-T cell therapies. Results: CD19/CD20-targeting GEM-DIMER molecules demonstrated binding to both CD19 and CD20 with affinities comparable to the individual parent antibodies. Importantly, CD19/CD20-targeting GEM-DIMER molecules demonstrated robust depletion of human B cells in overnight cultures of whole blood. Moreover, antibody-dependent cellular cytotoxicity (ADCC) was demonstrated in co-cultures of the human B cell lymphoma cell line Raji and human peripheral blood mononuclear cells (PBMC). As expected, due to the presence of dual Fc domains, CD19/CD20-targeting GEM-DIMER molecules exhibited enhanced binding to Fcgamma receptors, further explaining the robust effector functions observed. Conclusions: CD19/CD20-targeting GEM-DIMER molecules are promising candidates to provide efficient depletion of both CD19+ and CD20+ cells, providing potential for broad and deep depletion of B cells with reduced risk of emergence of antigen escape variants. These data support the advancement of CD19/CD20-targeting GEM-DIMER molecules in multiple indications where depletion of CD19+ and/or CD20+ B cells is needed. Preparations for clinical investigation are ongoing. Citation Format: Daniel J. Capon, Nelson L. Chan, Larisa Troitskaya, Marina Fomin, Ursula Edman, Brendon Frank, Benjamin Z. Capon, Brian Law, Steven J. Chapin, Gavin M. Lewis, Malcolm L. Gefter, Juha Punnonen. Beyond antibodies and CAR-T: Topologically-engineered, super-dimeric antibody-like molecules with dual Fc domains for trispecific, bivalent targeting of CD19, CD20, and Fcgamma receptors [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 2730.

Fludarabine Enhances Radiosensitivity by Promoting Ferroptosis in B-Cell Lymphoma.

The objective of this study is to investigate the impact of fludarabine, a signal transducer and activator of transcription-1 (STAT1) inhibitor, on the radiosensitivity of B-cell lymphoma (BCL) and to explore the underlying mechanisms. Radiotherapy is one of the primary treatments for BCL, and STAT1 plays a critical role in the transcription of cell proliferation-related genes, which are associated with radiotherapy and ferroptosis. This study aims to determine whether fludarabine can enhance the radiosensitivity of BCL and to elucidate the molecular pathways involved. Various in vitro methodologies, including CCK-8 assays, clonogenic formation assays, immunohistochemistry, immunofluorescence, flow cytometry, qRT-PCR, and Western blot analyses, were employed in B-cell lymphoma cell models to thoroughly investigate the effects of fludarabine on radiosensitivity. Subsequently, the obtained results were further validated through invivo animal models and by examining human diffuse large B-cell lymphoma (DLBCL) cancer samples. Our findings demonstrate that the combination of fludarabine and irradiation synergistically inhibits cell viability and colony formation, while inducing apoptosis and ferroptosis in B-cell lymphoma cell lines Raji and Su-DHL-10. Moreover, fludarabine was found to enhance the ferroptosis induced by radiation, thereby synergistically impeding the growth of BCL. In vivo experiments confirmed these findings, revealing that the intraperitoneal injection of fludarabine significantly enhanced the inhibitory effects of radiation on Raji cell xenograft models, leading to an increased percentage of ferroptosis compared to models without fludarabine. Additionally, the administration of liproxstatin-1, a ferroptosis inhibitor, attenuated the inhibition of xenograft growth caused by the combination of fludarabine and irradiation. Furthermore, our analysis of clinical data revealed that increased co-expression of STAT1 and GPX4 is associated with poor overall survival in patients with diffuse large B-cell lymphoma. These results highlight the potential of fludarabine to enhance radiosensitivity and ferroptosis induction as a promising therapeutic strategy for BCL. Our results demonstrated that fludarabine promoted radiation-induced BCL death through the ferroptosis pathway. We have identified a previously unrecognized mechanism in the fludarabine and radiation combination, indicating that it is necessary to conduct prospective clinical trials to verify this new treatment regimen in BCL.

TGF-β Inhibitor SD-208 Enhances the Anti-Tumor Efficacy of CD19 CAR-T Cells through a Reduction of CAR-Treg in Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia

Background: Acute lymphoblastic leukemia (ALL) is an incurable hematological malignancy with poor prognosis. Chimeric antigen receptor T cells (CAR-T) therapy is a milestone in the immunotherapy of relapsed or refractory B cells ALL (r/r B-ALL), but most cases are inevitably relapsed after CAR-T therapy and the causes are complex. Our preliminary work, a phase II clinical trial (#NCT02735291) involving pediatrics and adults with r/r B-ALL who were treated with CD19 CAR-T, exhibited that the overall survival (OS) and relapse-free survival (RFS) in patients with higher Treg are lower, and higher Treg may preclude the efficacy of CAR T therapy in r/r B-ALL. Further analysis of the frequencies of Treg within CD4 + cells at CD19 CAR-T therapy in r/r B-ALL patients investigated that higher circulating Treg, especially 1 week after CD19 CAR-T cells infusion, was a poor predict indicator for CD19 CAR-T therapy in r/r B-ALL patients. Methods: In the present study, we investigated the significance of CAR-Treg in CD19 CAR-T cells in clinical response and prognosis, and explored an inhibitive effect of TGF-β inhibitor SD-208 on the proliferation and lethality of CAR-Treg. Regulatory T cells (Treg) in CD19 CAR-T cells were tested in 34 patients with r/r B-ALL received CD19 CAR-T cells therapy, and then its significance on the complete remission (CR) rate, relapse-free survival (RFS) and overall survival (OS) was validated respectively. Next, TGF-β inhibitor SD-208 was added or not added in the culturing protocols of CD19 CAR-T cells, then the proportion of Treg and CAR-Treg as well as the anti-tumor ability of CD19 CAR-T cells were analyzed and compared in vitro. Results: 1) Higher proportion of Treg (≥7.2%) in CAR-T cells was a negative factor affecting RFS (p=0.0120) and OS (p=0.0162) in the r/r B-ALL patients with CAR-T therapy. 2) CD4 +CD25 +CD127 low/- Treg proliferated from 7.01% to 25.26% after anti-CD3 and anti-CD28 Abs activation. PBMCs cultured with SD-208 exhibited lower Treg levels than those of control groups (15.73% vs. 29.08%). 3) CD4 +CD25 +CD127 low/- Treg levels stimulated by SD-208 in CD19 CAR-T cells were lower than those in control groups both 24 and 48 hours (38.68% vs. 40.47% in 24 hours, 23.58% vs. 36.19% in 48 hours), and the treatment for 48 hours was most obvious. CAR +CD4 +CD25 +CD127 low/- CAR-Treg levels in CD19 CAR-T cells decreased by SD-208 stimulating compared with controls especially 48 hours (35.07% vs. 37.31% in 24 hours, 20.94% vs. 34.41% in 48 hours). The results exhibit that about 90% Treg in CD19 CAR-T cells are CAR-Treg, and inhibiting TGF-β by TGF-β/SMAD inhibitor SD-208 reduce the proliferation of Treg especially CAR-Treg in CD19 CAR-T cells. 4) To determine the function to CD19 CAR-T cell, LDH activity was utilized for tumor cell damage assay. Human Burkitt's lymphoma cell line Raji (CD19 +) was co-cultured with CD19 CAR-T cell for 24 hours before LDH assay. The percent of cytotoxicity calculated depending on the LDH activities showed that the inhibition rate of SD-208 treated CD19 CAR-T cell was significantly higher than that of control. To further determine the function of CD19 CAR-T cell Granzyme B and Perforin were tested. The results exhibited that SD-208 treated CD19 CAR-T cell had higher rates of Granzyme B and Perforin in CD3 +, CD3 +CD4 +, CD3 +CD8 + cells than controls, and achieved more functional. Conclusions: Inhibition of TGF-β by TGF-β/SMAD inhibitor SD-208 inhibit the proliferation of Treg in PBMCs of r/r B-ALL patients. TGF-β inhibitor SD-208 might improve the anti-tumor ability of CD19 CAR-T cells via decreasing Treg and CAR-Treg in r/r B-ALL.

Combination of CDK4/6 Inhibitor Palbociclib and PI3K Inhibitor Idelalisib Synergistically Induces an Anti-Tumor Effect in B-Cell Lymphoma and Overcomes Ibrutinib Resistance

Introduction: Aberrant activity of cyclin-dependent kinases 4/6 (CDK4/6) is frequently detected in B-cell non-Hodgkin's lymphoma (B-NHL), indicating the therapeutic implications of CDK4/6 inhibitors for B-cell malignancies. What's more, the introduction of agents targeting B cell receptor signaling, especially phosphatidylinositol 3-kinase (PI3K) isoform-specific inhibitors, is rapidly changing how B cell malignancies are treated. In this study, we aim to investigate whether the combination of CDK4/6 inhibitor palbociclib (PAL) and PI3Kδ inhibitor idelalisib (IDE) has synergistic anti-tumor effects in B cell lymphoma. Besides, we also evaluated the effect of co-administration PAL and IDE in Bruton's tyrosine kinase inhibitor (BTKi) resistant model. Methods: In vitro, cell viability was analyzed using Cell Titer-Glo ®️ Luminescent Cell Viability Assay. Flow cytometry was used to detect apoptosis and cell cycle arrest induction. Western Blotting analysis was used to detect the changes in essential proteins in related signaling pathways. We also conducted RNA-seq to evaluate the whole transcriptome changes brought by co-treatment with PAL and IDE. The synergistic anti-tumor effects of PAL and IDE were also evaluated in vivo. BTKi secondary resistant cell lines were generated by long-time exposure to ibrutinib and BTK C481S mutation cell line was conducted by lentivirus infection and puromycin selection. Results: Firstly, we analyzed diffuse large cell lymphoma (DLBCL) gene expression profile results from GEO database, and there were more than 50% patients carried cell cycle-related genes alterations, especially for CDKN2A-CDK4/6/CCND1-Rb machinery. Then, it was noteworthy that the high level of CDK4/6 mRNA and protein expression correlated with worse survival of DLBCL patients. Next, we illustrated that pharmacological inhibition of CDK4/6 kinase activity induced inhibition of cell proliferation, tumor cell apoptosis, and cell senescence. Meanwhile, PAL treatment upregulated the expression of PI3Kδ, p-AKT (S473), and p-mTOR (S2448), suggesting compensatory activation of the PI3K-AKT-mTOR signaling pathway. Thus, combination treatment of PAL and PI3K inhibitor IDE was assessed, and this combination synergistically induced a more potent anti-proliferative effect in several B-NHL cell lines, including mantle cell lymphoma cell lines (Z-138 and Jeko-1), DLBCL cell lines (U2932 and OCI-Ly8) and Burkitt lymphoma cell line (Raji). Meanwhile, co-administration treatment could also trigger synergistic anti-tumor activity in BTK inhibitor ibrutinib-resistant HBL-1 BTK C481S and HBL-1 IR cells (long time exposure to ibrutinib). Next, the flow cytometry results indicated that the B-NHL cells became apoptotic after the PAL and IDE treatment. The activation of apoptosis-related proteins (cleaved-caspase3 and cleaved-PARP) and decrease of anti-apoptosis proteins expression (BCL-2, BCL-xL, XIAP, and MCL-1) were induced by combination of CDK4/6 and PI3K inhibitor treatment. The combination of PAL and IDE also substantially increased the cell population of G0/G1 phase and elevated expression of CDK4, CDK6, and CyclinD1. Furthermore, the gene expression profile analysis demonstrated that the mRNA expression level of PLK1 was significantly decreased by co-administration treatment, rather than in PAL or IDE treatment alone. Finally, the combination treatment also showed synergistic anti-tumor activity in multiple tumor-bearing mice models. Conclusion: The combination of CDK4/6 inhibitor palbociclib and PI3K inhibitor idelalisib synergistically induced anti-tumor activity in B-cell lymphoma through downregulation of PLK1 expression, suggested a new combination direction for the treatment of B-NHL and even BTK inhibitor-resistant patients.

Identification of compounds that preferentially suppress the growth of T-cell acute lymphoblastic leukemia-derived cells.

T-cell acute lymphoblastic leukemia (T-ALL) is one of the most frequently occurring cancers in children and is associated with a poor prognosis. Here, we performed large-scale screening of natural compound libraries to identify potential drugs against T-ALL. We identified three low-molecular-weight compounds (auxarconjugatin-B, rumbrin, and lavendamycin) that inhibited the proliferation of the T-ALL cell line CCRF-CEM, but not that of the B lymphoma cell line Raji in a low concentration range. Among them, auxarconjugatin-B and rumbrin commonly contained a polyenyl 3-chloropyrrol in their chemical structure, therefore we chose auxarconjugatin-B for further analyses. Auxarconjugatin-B suppressed the in vitro growth of five human T-ALL cell lines and two T-ALL patient-derived cells, but not that of adult T-cell leukemia patient-derived cells. Cultured normal T cells were several-fold resistant to auxarconjugatin-B. Auxarconjugatin-B and its synthetic analogue Ra#37 depolarized the mitochondrial membrane potential of CCRF-CEM cells within 3 h of treatment. These compounds are promising seeds for developing novel anti-T-ALL drugs.

ArtinM Cytotoxicity in B Cells Derived from Non-Hodgkin's Lymphoma Depends on Syk and Src Family Kinases.

Receptors on the immune cell surface have a variety of glycans that may account for the immunomodulation induced by lectins, which have a carbohydrate recognition domain (CRD) that binds to monosaccharides or oligosaccharides in a specific manner. ArtinM, a D-mannose-binding lectin obtained from Artocarpus heterophyllus, has affinity for the N-glycans core. Immunomodulation by ArtinM toward the Th1 phenotype occurs via its interaction with TLR2/CD14 N-glycans on antigen-presenting cells, as well as recognition of CD3γ N-glycans on murine CD4+ and CD8+ T cells. ArtinM exerts a cytotoxic effect on Jurkat human leukemic T-cell line and human myeloid leukemia cell line (NB4). The current study evaluated the effects of ArtinM on murine and human B cells derived from non-Hodgkin’s lymphoma. We found that murine B cells are recognized by ArtinM via the CRD, and the ArtinM stimulus did not augment the proliferation rate or production of IL-2. However, murine B cell incubation with ArtinM augmented the rate of apoptosis, and this cytotoxic effect of ArtinM was also seen in human B cell-lines sourced from non-Hodgkin’s lymphoma Raji cell line. This cytotoxic effect was inhibited by the phosphatase activity of CD45 on Lck, and the protein kinases of the Src family contribute to cell death triggered by ArtinM.

Inhibition of PRMT5 Increases Sensitivity to BH3 Mimetics in Aggressive B Cell Malignancies

Introduction: Protein Arginine Methyltransferase 5 (PRMT5) is a type II methyltransferase that catalyzes symmetric dimethylation of arginine. PRMT5 is highly expressed in several types of aggressive B cell malignancies, and increased PRMT5 expression has been associated with poor disease prognosis. Several small molecule inhibitors targeting PRMT5 have been developed and showed promising anti-tumor effect in pre-clinical studies including in mantle cell lymphoma (MCL) and diffuse large B cell lymphoma (DLBCL). JNJ-64619178, a potent and specific PRMT5 inhibitor, exhibited broad antiproliferative activity in cancer cell lines from multiple cancer types and cell lines derived xenograft mouse model (Brehmer et al., 2021). In a phase 1 clinical trial of JNJ-64619178 (NCT03573310), patients with advanced solid tumors and non-Hodgkin lymphoma demonstrated manageable toxicity and preliminary evidence of antitumor activity at selected dose levels (Villar et al., 2020). In addition, in patients with lower-risk myelodysplastic syndromes, JNJ-64619178 had manageable hematologic toxicity at the selected expansion dose, but did not have significant clinical activity (Haque et al., 2021). Inhibition of PRMT5 alone could induce cell cycle arrest instead of apoptosis depending on the types of cancer (Zhu et al., 2019), therefore we hypothesized that the combination of PRMT5i and pro-apoptotic BH3 mimetics therapies could be synergistic in aggressive B cell malignancies. Methods: We used dynamic BH3 profiling (DBP) to measure the net pro-apoptotic signaling induced by ex vivo drug treatments (as per Montero et al., 2015). Other standard techniques included shRNA mediated gene knockdown, Western blot, cell viability assays including PI/Annexin V and CellTiter-Glo, and reactive oxygen species (ROS) measurement by flow cytometry with dihydroethidium (DHE) staining. The DLBCL cell lines (HBL1, TMD8, RI-1, OCI-Ly1, Karpas 422, SUDHL4, Toledo), double hit lymphoma (DHL) patient-derived xenograft (PDX) cell lines (DW19), mantle cell lymphoma (MCL) cell lines (Mino, Jeko-1), T cell acute leukemia (T-ALL) cell line (Jurkat) and Burkitt lymphoma (BL) cell line (Raji) were used to investigate the in vitro anti-cancer activity of JNJ-64619178 (Janssen Pharmaceuticals) and BH3 mimetics (venetoclax [BCL2i], S63845 [MCL-1i] and A1331852 [BCL-xLi], Selleckchem). SynergyFinder was used to calculate drug synergism. Results: In all 7 DLBCL cell lines and the DHL-PDX cell line, we found synergistic induction of apoptosis with JNJ-64619178 plus venetoclax (synergy score, 2.82~51.42). The lowest level of synergy was observed in OCI-Ly1 cells, which are intrinsically highly sensitive to venetoclax monotherapy. The combination of JNJ-64619178 with S63845 synergistically induced apoptosis in 5 out of 7 cell lines (synergy score, -1.91~35.64). While the combination of JNJ-64619178 with A1331852 synergistically induced apoptosis in all 7 cell lines (synergy score 1.88~46.73), 4 of them had weak synergy score (1.88~9.4). To confirm the effects we observed with pharmacologic inhibition of PRMT5, we performed shRNA mediated knockdown of PRMT5, which similarly revealed increased sensitivity to venetoclax. In MCL cell lines, combining JNJ-64619178 with venetoclax or A1331852 was more synergistic in inducing cell death than combining with S63845. The T-ALL Jurkat cell line was resistant to JNJ-64619178 and single BH3 mimetic-induced apoptosis. Combining JNJ-64619178 with A1331852 but not venetoclax or S63845 strongly induced apoptosis. The Burkitt cell line Raji was resistant to JNJ-64619178, venetoclax and A1331852, but was sensitive to S63845. The addition of JNJ-64619178 and a single BH3 mimetic in these Raji cells had only a minor synergistic effect. The synergy scores are summarized in Figure 1. Using DBP, JNJ-64619178 was found to increase overall apoptotic priming of DLBCL and MCL cell lines (> 15% increase in cytochrome c loss). By measuring ROS levels, we found increased ROS production (> 2-fold increase in median fluorescent intensity) in response to JNJ-64619178 before the onset of apoptosis, suggesting a possible mechanism by which JNJ-64619178 increases apoptotic priming. Conclusions: The combination of a PRMT5 inhibitor with BH3 mimetics, especially venetoclax, is worthy of further exploration as a potential therapeutic strategy for DLBCL and MCL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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.
 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.
 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 < 0.05).
 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.

Surface-Functionalized Polymeric siRNA Nanoparticles for Tunable Targeting and Intracellular Delivery to Hematologic Cancer Cells.

To date, the application of RNA therapeutics to hematologic malignancies has been challenging owing to the resistance of blood cancer cells against conventional transfection methods. Herein, triple-targeting moiety-functionalized polymeric small interfering RNA (siRNA) nanoparticles were systematically developed for efficient targeted delivery of RNA therapeutics to hematologic cancer cells. Polymeric siRNAs were synthesized using rolling circle transcription and were surface-functionalized with three types of targeting moieties─a natural ligand and two additional combinations of cell-specific antibodies─for tunable targetability. As a proof of concept, the optimization of the hyaluronic acid/antibody conjugation ratio was performed for selective intracellular delivery to various non-Hodgkin's lymphoma (NHL) cell lines (Daudi, Raji, Ramos, and Toledo cells) via receptor-mediated endocytosis. The engineered nanoparticles showed almost 10-fold enhanced NHL-specific intracellular delivery and induced significant in vitro anticancer effects. This multitargeted nanoparticle platform may effectively support the intracellular delivery of polymeric siRNA sequences, and thus promote therapeutic effects in hematopoietic malignancies.

Discovery of novel ibrutinib analogues to treat malignant melanoma

A series of novel ibrutinib analogues was synthesized, and their proliferation inhibitory activities against various B lymphoma cell lines (DaudiB and Raji) and solid tumor cells (B16, CT26, HepG2 and 4T1) were evaluated. The most potent compound, YL7, exhibited strong antiproliferative activity in all cell lines, and its IC50 value in B16 cells was almost 9-fold better than that of ibrutinib. Mechanism of action studies showed that YL7 inhibited proliferation and migration and induced G1 cell cycle arrest, apoptosis and autophagy in B16 cells. Further assessment of in vivo antitumor efficacies demonstrated that YL7 significantly inhibited the growth of B16 melanoma. These preliminary studies suggest that it is reasonable to modify the structure of ibrutinib for antimelanoma treatment.

Nanobody-armed T cells endow CAR-T cells with cytotoxicity against lymphoma cells

BackgroundTaking advantage of nanobodies (Nbs) in immunotherapy, we investigated the cytotoxicity of Nb-based chimeric antigen receptor T cells (Nb CAR-T) against lymphoma cells.MethodsCD19 Nb CAR-T, CD20 Nb CAR-T, and Bispecific Nb CAR-T cells were generated by panning anti-human CD19- and CD20-specific nanobody sequences from a natural Nb-expressing phage display library, integrating Nb genes with a lentiviral cassette that included other CAR elements, and finally transducing T cells that were expanded under an optimization system with the above generated CAR lentivirus. Prepared Nb CAR-T cells were cocultured with tumour cell lines or primary tumour cells for 24 h or 5 days to evaluate their biological function.ResultsThe nanobodies that we selected from the natural Nb-expressing phage display library had a high affinity and specificity for CD19 and CD20. CD19 Nb CAR-T, CD20 Nb CAR-T and Bispecific Nb CAR-T cells were successfully constructed, and these Nb CAR-T cells could strongly recognize Burkitt lymphoma cell lines (Raji and Daudi), thereby leading to activation, enhanced proliferation, and specific killing of target cells. Furthermore, similar results were obtained when using patient samples as target cells, with a cytotoxicity of approximately 60%.ConclusionsNanobody-based CAR-T cells can kill both tumour cell lines and patient-derived tumour cells in vitro, and Nb-based CAR-T cells may be a promising therapeutic strategy in future immunotherapy.

Upregulation of CD38 Expression on Multiple Myeloma Cells By the HDAC Inhibitor Ricolinostat Augments the Efficacy of Daratumumab

Background:The anti-CD38 antibody daratumumab is effective and increasingly being utilized to treat multiple myeloma. A clinical observation with daratumumab is that expression of CD38 on pretreatment myeloma cells correlates with efficacy, and patients with higher CD38 expression have a higher chance to respond. Moreover, during daratumumab therapy, there are reduced CD38 expression levels on myeloma cells, favoring immune escape and disease progression. We have recently shown that the histone deacetylase (HDAC) inhibitor panobinostat induces CD38 upregulation and augments the antibody-dependent cellular cytotoxicity (ADCC) of daratumumab (García-Guerrero E, Blood 2017). Here, we investigated the alternative HDAC inhibitor ricolinostat and all-trans retinoic acid (ATRA) to determine whether upregulation of CD38 is a class effect, and to identify the substance that works most synergistically with daratumumab.Methods:The myeloma cell lines MM1.S and OPM2 were treated with titrated doses of ricolinostat (0, 5, 10 µM), ATRA (0, 10, 25 nM) and panobinostat (0, 10, 25 nM). Expression of CD38, B-cell maturation antigen (BCMA) and SLAMF7 was analyzed by flow cytometry after 24, 48 and 72 h of exposure. ADCC against HDAC inhibitor-treated and untreated myeloma cells was analyzed after addition of PBMC at an effector to target ratio of 25:1 in the presence of daratumumab (1, 10, 50 ug/mL) or isotype control.Results:We first treated the myeloma cell line MM1.S with each of the HDAC inhibitors and analyzed their direct cytotoxic antimyeloma effect. We found the strongest effect with ricolinostat where live MM1.S myeloma cells had decreased to 50% and 20% after 48 h of exposure to 5 and 10 µM of the drug, respectively. Consistent with previous work the direct anti-myeloma effect of panobinostat (decrease of live MM1.S myeloma cells to 85% and 50% after 48 h of exposure to 10 and 25 nM, respectively) and ATRA (no cytotoxic effect) was lower compared to ricolinostat.We then analyzed expression of CD38 on residual live i.e. 7-AAD negative MM1.S cells by flow cytometry and observed a 2-fold (5 µM) and 2,5-fold (10 µM) increase in CD38 expression by mean fluorescence intensity (MFI) after treatment with ricolinostat-treated compared to untreated MM1.S cells. The increase in CD38 expression was already detectable after 24 h and further increased with longer exposure to the drug. Off note, withdrawal of ricolinostat resulted in rapid decline of CD38 expression to baseline levels but increased again to the same magnitude within 24 h of re-exposure to the drug. Overall, the increase in CD38 expression was strongest with ricolinostat followed by panobinostat and ATRA, which we confirmed with several myeloma cell lines and primary myeloma cells. We also confirmed that ricolinostat-induced upregulation of CD38 specifically occurred in myeloma cells and not in the lymphoma cell lines Raji and JeKo-1. In contrast to CD38, expression of BCMA and SLAMF7 after treatment of myeloma cells with ricolinostat had decreased.We then treated myeloma cells with ricolinostat for 48 hours to induce upregulation of CD38 and then performed ADCC assays with daratumumab in the presence of ricolinostat. We found, that the combination treatment ricolinostat plus daratumumab eliminated 95,6% of MM1.S cells after 20h; while 87,4% were eliminated with ricolinostat alone, and only 22,6% with daratumumab alone. Preliminary data show that ricolinostat does not affect expression of the complement inhibitor CD55 and induces a lower increase in CD59 expression compared to panobinostat. Experiments to evaluate complement-dependent cytotoxicity (CDC), and to confirm the synergistic antimyeloma effect of ricolinostat and daratumumab in a murine xenograft myeloma model, are ongoing.Conclusions:Our data demonstrate that the HDAC inhibitor ricolinostat induces upregulation of CD38 expression on myeloma cells. The increase in CD38 expression after treatment with ricolinostat is stronger compared to panobinostat and ATRA. Our data suggest that upregulation of CD38 on myeloma cells is a class effect of HDAC inhibitors, and identify ricolinostat as a highly synergistic combination partner for daratumumab, due to its considerable intrinsic antimyeloma activity. Our data suggest that combination treatment with ricolinostat and daratumumab could increase response rates and extend duration of responses of daratumumab therapy. DisclosuresNo relevant conflicts of interest to declare.

Targeted alpha immunotherapy of CD20-positive B-cell lymphoma model: dosimetry estimate of 225Ac-DOTA-rituximab using 64Cu-DOTA-rituximab.

The aim of this study was to evaluate the radiation dosimetry of alpha-emitter 225Ac-DOTA-rituximab using Monte Carlo simulation of 64Cu-DOTA-rituximab. CD20 expression was evaluated in lymphoma cell lines (Jurkat and Raji). DOTA-rituximab was conjugated and then chelated by 64Cu. Tumor xenograft models were established in BALB/c-nu mice. Animal PET/CT imaging was obtained after tail vein injection with and without a pre-dose of 2mg of cold rituximab. Specific binding of tumors was evaluated by an organ distribution assay and autoradiography. CD20 expression in tumor tissues was evaluated by immunohistochemistry. The residence time was calculated using 64Cu-DOTA-rituximab PET/CT acquisition data using OLINDA/EXM software. 225Ac-DOTA-rituximab tumor dosimetry was performed using Monte Carlo simulation with 64Cu-DOTA-rituximab PET/CT images. Specific binding of Raji cells (CD20 positive) was 90 times that of Jurkat cells (CD20 negative) (p < 0.0001). Immunoreactivity was more than 75%. PET/CT imaging with 64Cu-DOTA-rituximab was specifically observed in tumors. The radioactivity of the tumor was much higher than that of other organs, and tumor uptake was related to CD20 expression. The predicted human dose for the administration of 64Cu-DOTA-rituximab was measured as the effective dose (1.07E-02mSv/MBq). In the tumor region, equivalent doses of 225Ac-DOTA-rituximab (14 SvRBE5/MBq) were much higher (74-fold) than those of 64Cu-DOTA-rituximab (0.19 SvRBE5/MBq) (p < 0.01). Tumor dosimetry of 225Ac-DOTA-rituximab can be estimated via the Monte Carlo simulation of 64Cu-DOTA-rituximab. 225Ac-DOTA-rituximab can be employed for lymphoma as targeted alpha therapy.

Chromosomal Translocation t(11;14) Induced By the Cre-Loxp System in Normal B Cell-Derived Ips Cells for the Study of Myeloma-Initiating Cells

The cellular origin of multiple myeloma (MM) has not yet been identified. Based on immunoglobulin heavy chain (IgH) gene analysis, myeloma cells are derived from mature B cells. Chromosomal aberrations such as trisomy and chromosomal translocation (cTr) play a critical role in the early tumorigenesis of MM. We hypothesized that the abnormal cells from which myeloma cells originate might be mature B lymphocytes with chromosomal or genetic changes in the reprogrammed state that enable them to acquire the potential to become tumors in the process of redifferentiation into B lymphocytes. We established induced pluripotent stem cells (iPSs) from normal B lymphocytes (BiPSCs: BiPSC13 &amp; MIB2-6); these BiPSCs have the same VDJ rearrangement of IgH as the original B lymphocytes and differentiate into CD34+/CD38- hematopoietic progenitor cells co-culture with stromal cells, AGM-S3 (Sci Rep, 2017). We then established a method to induce reciprocal cTr t(11;14), which is a reciprocal cTr between IgH and CCND1 and the most frequent cTr in MM, using the CRISPR/Cas9 system; cTr was induced by infection of IgH-CCND1 lentiCRISPRv2 lentivirus, which targets the human IgH Eµ region and 13kb upstream of the CCND1 coding sequence, to BiPSCs (Oncol Lett, 2019). Subsequently, we established cell lines carrying reciprocal cTr t(11;14) between CCND1 and either an allele in which VDJ rearrangement of IgH had been completed or an allele in which VDJ rearrangement had not been completed (stopped at DJ joining) in BiPSC13 t(11;14) (AZ &amp; AX) and MIB2-6 t(11;14) (BC &amp; BG), respectively. These BiPSCs differentiated into CD34+/CD38-/CD45+/-/CD43+/- hematopoietic progenitors cells in co-culture with AGM-S3 or in stem cell differentiation medium; this was subsequently confirmed by the differentiation into granulocytes, macrophages, and erythroblasts in a colony-formation assay. We are now trying to produce BiPSCs in which cTr t(11;14) is induced when they differentiate into mature B cells expressing CD27. First, we used the Cre-loxP recombination system to induce cTr t(11;14) in BiPSCs. BiPSCs were transfected with IgH loxP-Neo-loxP knock-in vector and IgH lentiCRISPRv2 vector. Subsequently, G418-resistant BiPSCs carrying loxP-Neo-loxP in IgH were transfected with iCre-EGFP. After removing the loxP-Neo site from EGFP-positive cells, BiPSCs carrying IgH-loxP were transfected with CCND1 loxP-FRT3-Neo-FRT3 knock-in vector and CCND1 lentiCRISPRv2 vector. Subsequently, G418-resistant BiPSCs carrying IgH-loxP in IgH and loxP-FRT3-Neo-FRT3 in CCND1 were transfected with Flpo-EGFP. After removing the FRT3-Neo site from EGFP-positive cells, BiPSCs carrying IgH-loxP in IgH and CCND1-loxP-FRT3 in CCND1 were transfected with iCre-HygR. Hygromycin B-resistant cells were picked, the reciprocal cTr t(11;14) was confirmed by polymerase chain reaction, and we established BiPSCs with der(11)t(11;14) and BiPSCs with der(14)t(11;14). We also developed a system in which Cre is expressed along with CD27 expression in the B cell lymphoma cell line Raji. These BiPSCs could be useful for the study of myeloma-initiating cells, but whether they would be able to be redifferentiated into B lymphocyte is important. Disclosures Hanamura: Mundipharma K.K.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MSD K.K.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sanofi K.K.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SHIONOGI Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis Pharma K.K.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; DAIICHI SANKYO COMPANY, LIMITED: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kyowa Kirin Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Eisai Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; NIPPON SHINYAKU CO.,LTD.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer Japan Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda Pharmaceutical Company Limited: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen Pharmaceutical K.K.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Ono Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Abstract 2216: Combinational strategy targeting B cell malignancy using iPSC engineered CAR-NK (FT596) and CAR-T cell (FT819) platforms with therapeutic antibody to achieve an effective deep and durable response

Abstract The use of induced pluripotent stem cells (iPSCs) to derive immune effector cells offers distinct advantages for immune therapy over existing patient- or donor- derived platforms, not only in terms of scalable manufacturing and precision genetic engineering at the clonal level, but also in allowing the generation and combinational use of multiple effector cell types each with distinct characteristics. Taking cues from the natural propagation of innate to adaptive effector responses, here we describe the combined use of multi-engineered iPSC derived Natural Killer (iNK) and T (iT) cell platforms in order to exploit the unique properties of each cell type to achieve both depth and durability of response for hematological malignancies. As innate cells, NK cells are characterized by the capacity for spontaneous reactivity, either in response to cell surface antigen or downregulation of class I MHC, and the rapid kinetic under which NK cells operate make them an ideal candidate to achieve a depth of response that outpaces the limiting effects of cytokine support. FT596 was developed as a dual-targeted iNK cell platform engineered to express both a CD19-directed, NK cell-optimized (NKG2D-2B4-CD3ζ) chimeric antigen receptor (CAR) and a high-affinity, non-cleavable Fc receptor (hnCD16), enabling multi-targeting through combination with therapeutic antibodies. The activity of each receptor is further enhanced by the expression of an IL15-IL15Ra fusion receptor, which also allows the cells to expand in the absence of exogenous cytokine support and prolongs cell survival in vitro and in vivo. T cells are exquisitely specific and undergo rapid clonal expansion and differentiation in response to target antigen, and antigen driven persistence has been demonstrated as a key determinant in efficacy in primary CAR-T cell immune therapy. FT819 is an iT cell platform engineered to express a functionally optimized CD19-CAR (1XX) that has been genome edited into the T cell receptor (TCR) alpha constant (TRAC) locus to provide ideal CAR activity and to prevent TCR expression, thereby avoiding the complications of GVH reactivity in an allogeneic setting. In vivo, both FT596 and FT819 showed stable levels of tumor cell clearance against the CD19+ acute lymphoblastic leukemia cell line NALM6, comparable to that of primary CAR19 T cells (p*&amp;lt;0.0001 for FT596, FT819 or Primary CART vs NALM6 alone). FT596 also shows enhanced clearance of CD19+CD20+ Burkitts lymphoma cell line RAJI when used in combination with rituximab (p=0.0002 vs rituximab alone). Collectively, these studies suggest a compounded anti-tumor effect can be achieved utilizing the inherent properties of engineered CAR-iNK cells together with therapeutic antibody combined with engineered CAR-iT cells which will be highlighted in this presentation. Citation Format: Jode P. Goodridge, John W. Reiser, Ryan Bjordahl, Milli Mandal, Chia-wei Chang, Raedun Clarck, Sajid Mahmood, Huang Zhu, Svetlana Gaidarova, Robert Blum, Frank Cichocki, Hui-ting Hsu, Greg Bonello, Tom Lee, Brian Groff, Karl-Johan Mamlberg, Bruce Walcheck, Jeffrey S. Miller, Dan Kaufman, Bahram Valamehr. Combinational strategy targeting B cell malignancy using iPSC engineered CAR-NK (FT596) and CAR-T cell (FT819) platforms with therapeutic antibody to achieve an effective deep and durable response [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2216.

Effect of carboplatin injection on Bcl-2 protein expression and apoptosis induction in Raji cells.

To investigate the effects of carboplatin (CBP) injection on apoptosis induction in the human lymphoma cell line Raji and to explore the underlying mechanism, Raji cells were randomly divided into two treatment groups. Cells in the experimental groups were treated with 15 μM CBP injection, those in the control groups were treated with solvent, and both groups were treated for 24, 48 and 72 h. Cells from each group were collected for subsequent assays. For each group, the relative expression of B-cell lymphoma-2 (Bcl-2) was determined by Western blot (WB), the expression pattern of Bcl-2 was observed by immunocytochemistry (ICC), and cell apoptosis was observed after Hoechst 33342 staining. Real-time PCR detection of the relative expression levels of the Bax and caspase-3 genes in each group of cells were performed. The WB results showed that the relative expression of the Bcl-2 protein significantly decreased 48 and 72 h after treatment in the CBP groups compared with the control groups (P<0.001), and a significant decrease in the expression of this protein was also noted at 48 h vs 24 h, 72 h vs 48 h, and 72 h vs 24 h with extremely significant differences (P<0.001). Moreover, the expression of the Bcl-2 protein decreased as the duration of CBP treatment increased, showing a time-dependent manner. The ICC results were consistent with the WB findings. The expression of the Bcl-2 protein in the CBP treatment group was significantly reduced 48 h and 72 h after treatment compared with the control group (P<0.001). A time-dependent manner was also noted in the expression of this protein, i.e., the expression level decreased gradually at 24, 48, and 72 h after treatment with statistically significant differences (P<0.001). Hoechst 33342 staining showed that the apoptosis rates at the three time points in the treatment groups were significantly higher than those in the control groups (P<0.001), and a time-effect relationship was observed. The apoptosis rate increased over time with a significant difference (P<0.05). The PCR results showed that the Bax and caspase-3 gene expression trend was the same but opposite that of Bcl-2. After treatment for 24 h and 48 h, the gene expression of the medication groups decreased with a very significant difference (P<0.001), and with prolonged action time, the relative expression of the genes in the medication groups showed an upward trend. Comparing 48 h with 72 h and 24 h with 72 h, the gene expression levels also increased, reaching a very significant difference (P<0.001), and there was a certain time dependence. CBP injection significantly reduced the expression of the Bcl-2 protein and induced apoptosis of Raji cells in a time-dependent manner. Moreover, CBP injection can increase the expression levels of the Bax and caspase-3 genes.

Characterization of resistance to a recombinant hexameric Fas-ligand (APO010) in human cancer cell lines

Multiple myeloma remains a hard-to-treat cancer as all patients eventually progress because of drug resistance. Thus, there is a need for novel and non-cross-resistant treatment options, and we aimed to address this issue by introducing a new immuno-oncology drug (APO010) in multiple myeloma treatment. APO010 is a hexameric Fas-ligand that mimics cytotoxic T-lymphocyte signaling through the Fas-receptor to induce apoptosis. APO010 is currently in clinical trials with multiple myeloma patients. Thus, an understanding of the mechanisms contributing to resistance to APO010 will be essential for future clinical studies with APO010, and it might be possible to develop strategies to circumvent this resistance. We developed APO010-resistant variants of human multiple myeloma cell lines (LP1, MOLP-8, and KMS-12-BM) and a human Burkitt's lymphoma cell line (Raji) by exposing the cells to gradually increasing concentrations of APO010 over a period of 6-12 months. The resistant cell lines were characterized on the basis of immunocytochemistry, Fas-receptor protein expression, mRNA expression analysis, and pathway analysis. APO010-resistant cell lines exhibited a 4- to 520-fold increase in resistance to APO010 and still remained sensitive to other chemotherapeutics. Downregulation of the Fas-receptor protein expression was observed in all resistant cell lines. mRNA expression analysis of the resistant versus parental cell lines confirmed a significant alteration in FAS expression between sensitive and resistant cell lines (p = 0.03), while pathway analysis revealed alterations in mRNA signaling pathways of Fas. On the basis of the pre-clinical data obtained, it can be concluded that downregulation of Fas-receptor can mediate resistance to APO010.

FT596: Translation of First-of-Kind Multi-Antigen Targeted Off-the-Shelf CAR-NK Cell with Engineered Persistence for the Treatment of B Cell Malignancies

Induced pluripotent stem cell (iPSC)-derived effector cells offer distinct advantages for immune therapy over existing patient- or donor- derived platforms, both in terms of scalable manufacturing from a renewable starting cellular material and precision genetic engineering that is performed at the single-cell level. iPSC derived natural killer (iNK) cells offer the further advantage of innate reactivity to stress ligands and MHC downregulation and the potential to recruit downstream adaptive responses. These unique features form the basis of our multi-antigen targeted chimeric antigen receptor (CAR) CAR-iNK cell product candidate, termed FT596, which is further combined with additional functionality to enhance effector function. FT596 is consistently manufactured from a master iPSC line engineered to uniformly express an NK cell-calibrated CD19-targeting CAR (CD19-CAR), an enhanced functioning high-affinity, non-cleavable CD16 (hnCD16) and a recombinant fusion of IL-15 and IL-15 receptor alpha (IL-15RF) for cytokine-autonomous persistence. The design of the CD19-CAR involved exploiting the intrinsic polyfunctionality of NK cells, which function by engaging multiple signaling pathways activated through combinations of distinct germline encoded receptors. Using this approach, the transmembrane region of activating receptor NKG2D, combined with the intracellular signaling domains of SLAM co-receptor 2B4 and CD3ζ, proved the most effective in triggering antigen specific functional responses in NK cells. Chimerization of an anti-CD19 scFv onto this NKG2D-2B4-CD3ζ signaling platform produced specific in vitro recognition of CD19+ B cell lymphoma cells in short-term and long-term NK cytotoxicity assays (&amp;gt;80% and &amp;lt;40% clearance of tumor cells at 60H, p&amp;lt;0.001 respectively). The functionality of the CD19-CAR was further enhanced in combination with autonomous IL-15 signaling. Introduction of the IL-15RF enabled expansion of iNK cells without addition of soluble cytokine and greatly improved longevity and functional persistence of iNK cells both in vitro and in animal models. Moreover, iNK cells modified with IL-15RF showed enhanced functional maturation, including upregulated expression of effector molecules such as granzyme B. iNK cells with both CD19-CAR and IL-15RF resulted in enhanced CAR functionality in vitro, and mouse models for B cell malignancy demonstrated that treatment with iNK cells engineered with CD19-CAR and IL-15RF were curative against B cell lymphoma (p&amp;lt;0.002), when compared with iNK cells alone or iNK cells modified with CD19-CAR alone. In combination with hnCD16, co-expression of CD19-CAR and IL15-RF culminates in iNK cells capable of dual-specificity through combinatorial use with monoclonal antibodies to tackle antigen escape. In long term killing assays, FT596 alone demonstrated equivalent levels of CD19 targeted anti-tumor activity as primary CD19-targeted CAR (CAR19) T cells when tested against CD19+ CD20+ B lymphoblast target cells and demonstrated enhanced levels of activity when used in combination with anti-CD20 (rituximab). When targeting CD19- CD20+ B lymphoblast target cells and used in combination with rituximab, only FT596 was able to effectively eliminate the CD19 antigen escaped target cell (64% vs 30% clearance of tumor cells at 36H vs rituximab alone). In vivo FT596 showed equivalent levels of tumor cell clearance as primary CAR19 T cells against the CD19+ acute lymphoblastic leukemia cell line NALM6 and CD19+CD20+ Burkitts lymphoma cell line RAJI, and enhanced clearance of RAJI tumor cells in combination with rituximab (p=0.0002). Furthermore, utilizing an allogenic human CD34 engrafted NSG mouse model, FT596 demonstrated improved survival and safety over primary CAR19 T cells, either as a monotherapy or as a combination therapy with rituximab versus RAJI tumor cells. Together, these studies demonstrate FT596 provides a multi-antigen targeting, potent and persistent engineered immune cell that is derived from a master iPSC line which utilizes the intrinsic versatility of NK cells to enable a highly effective combination therapy in a single, standardized, scalable, off-the-shelf platform and supports the rational for a first-of-kind Phase I Study as a monotherapy and in combination with CD20-targeted mAbs including rituximab in subjects with relapsed/refractory B-cell lymphoma and leukemia. Figure Disclosures Goodridge: FATE THERAPEUTICS: Employment. Mahmood:Fate Therapeutics, Inc: Employment. Gaidarova:Fate Therapeutics, Inc: Employment. Bjordahl:Fate Therapeutics, Inc.: Employment. Cichocki:Fate Therapeutics, Inc: Research Funding. Chu:FATE THERAPEUTICS: Employment. Bonello:Fate Therapeutics, Inc.: Employment. Lee:Fate Therapeutics, Inc.: Employment. Groff:FATE THERAPEUTICS: Employment. Meza:FATE THERAPEUTICS: Employment. Malmberg:Vycellix: Consultancy, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc.: Consultancy, Research Funding. Miller:Moderna: Membership on an entity's Board of Directors or advisory committees; Dr. Reddys Laboratory: Membership on an entity's Board of Directors or advisory committees; CytoSen: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc: Consultancy, Research Funding; OnKImmune: Membership on an entity's Board of Directors or advisory committees; GT BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Kaufman:FATE Therapeutics: Consultancy, Research Funding. Valamehr:Fate Therapeutics, Inc: Employment.