Solid Tumor Cell Lines Research Articles

Anti-Tumor Effects of a Penetratin Peptide Targeting Transcription of E2F-1, 2 and 3a Is Enhanced When Used in Combination with Pemetrexed or Cisplatin.

Simple SummaryThe E2F family of transcription factors are essential for cell proliferation, differentiation, and DNA repair. They are commonly overexpressed or dysregulated in cancer as a consequence of inactivation or mutations in the retinoblastoma protein. Therefore, one or more of the activating E2Fs (E2F-1, 2, and 3a) have been recognized as antitumor targets. The combination of a peptide targeting transcription of E2F-1, 2, and 3a, with cisplatin, and especially with pemetrexed, showed enhanced antitumor activity in-vitro and in-vivo and has promise for the treatment of patients with various tumors, and in particular, lung adenocarcinoma.Background: We tested the antitumor effects of a modified E2F peptide substituting D-Arg for L-Arg, conjugated to penetratin (PEP) against solid tumor cell lines and the CCRF-leukemia cell line, alone and in combination with pemetrexed or with cisplatin. For in-vivo studies, the peptide was encapsulated in PEGylated liposomes (PL-PEP) to increase half-life and stability. Methods: Prostate cancer (DU145 and PC3), breast cancer (MCF7, MDA-MB-468, and 4T1), lymphoma (CCRF-CEM), and non-small cell lung cancer (NSCLC) cell lines (H2009, H441, H1975, and H2228) were treated with D-Arg PEP in combination with cisplatin or pemetrexed. Western blot analysis was performed on the NSCLC for E2F-1, pRb, thymidylate synthase, and thymidine kinase. The H2009 cell line was selected for an in-vivo study. Results: When the PEP was combined with cisplatin and tested against solid tumor cell lines and the CCRF-CEM leukemia cell line, there was a modest synergistic effect. A marked synergistic effect was seen when the combination of pemetrexed and the PEP was tested against the adenocarcinoma lung cancer cell lines. The addition of the PEP to pemetrexed enhanced the antitumor effects of pemetrexed in a xenograft of the H2009 in mice. Conclusions: The D-Arg PEP in combination with cisplatin caused synergistic cell kill against prostate, breast, lung cancers, and the CCRF-CEM cell line. Marked synergy resulted when the D-Arg PEP was used in combination with pemetrexed against the lung adenocarcinoma cell lines. A xenograft study using the PL-PEP in combination with pemetrexed showed enhanced anti-tumor effects compared to each drug alone.

Late‐stage Rh(II)‐catalyzed Nitrene Transfer for the Synthesis of Guaianolide Analogs with Enhanced Antiproliferative Activity

AbstractA set of new guaianolide derivatives (1–9) was obtained from ludartin, achalensolide, and 11,13‐dihydroachalensolide by application of catalytic nitrene transfer reactions. Intermolecular nitrene C(sp3)−H insertions led to the amination of C‐1, C‐2, and C‐10 positions, while alkene aziridination was also observed under these reaction conditions. The antiproliferative activity of natural compounds and their derivatives was evaluated against a panel of human solid tumor cell lines. The results show that an increase in the biological activity was observed following amination at the C‐2 position of Ludartin, thereby demonstrating the interest in late‐stage C−H amination to improve the bioactivity of natural products.

The antitumor capacity of mesothelin-CAR-T cells in targeting solid tumors in mice

Since the approval of chimeric antigen receptor (CAR) T cell therapy targeting CD19 by the FDA, CAR-T cell therapy has received increasing attention as a new method for targeting tumors. Although CAR-T cell therapy has a good effect against hematological malignancies, it has been less effective against solid tumors. In the present study, we selected mesothelin (MSLN/MESO) as a target for CAR-T cells because it is highly expressed by solid tumors but only expressed at low levels by normal tissues. We engineered a third generation MSLN-CAR comprising a single-chain variable fragment (scFv) targeting MSLN (MSLN-scFv), a CD8 transmembrane domain, the costimulatory domains from CD28 and 4-1BB, and the activating domain CD3ζ. In vitro, MSLN-CAR-T cells killed various solid tumor cell lines, demonstrating that it could specifically kill MSLN-positive cells and release cytokines. In vivo, we investigated the effects of MSLN-CAR-T cell therapy against ovarian, breast, and colorectal cancer cell-line-derived xenografts (CDX) and MSLN-positive colorectal and gastric cancer patient-derived xenografts (PDX). MSLN-CAR decreased the growth of MSLN-positive tumors concomitant with significantly increased T cells and cytokine levels compared to the control group. These results indicated that modified MSLN-CAR-T cells could be a promising therapeutic approach for solid tumors.

Analysis of Side Population in Solid Tumor Cell Lines

Analysis of Side Population in Solid Tumor Cell Lines

Analysis of Side Population in Solid Tumor Cell Lines

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great significance for tumor research. Currently, several techniques are used for the identification and purification of CSCs from tumor tissues and tumor cell lines. Separation and analysis of side population (SP) cells are two of the commonly used methods. The methods rely on the ability of CSCs to rapidly expel fluorescent dyes, such as Hoechst 33342. The efflux of the dye is associated with the ATP-binding cassette (ABC) transporters and can be inhibited by ABC transporter inhibitors. Methods for staining cultured tumor cells with Hoechst 33342 and analyzing the proportion of their SP cells by flow cytometry are described. This assay is convenient, fast, and cost-effective. Data generated in this assay can contribute to a better understanding of the effect of genes or other extracellular and intracellular signals on the stemness properties of tumor cells.

Structure-activity relationship and mechanistic studies for a series of cinnamyl hydroxamate histone deacetylase inhibitors

Histone deacetylases (HDACs) are a family of enzymes that modulate the acetylation status histones and non-histone proteins. Histone deacetylase inhibitors (HDACis) have emerged as an alternative therapeutic approach for the treatment of several malignancies. Herein, a series of urea-based cinnamyl hydroxamate derivatives is presented as potential anticancer HDACis. In addition, structure–activity relationship (SAR) studies have been performed in order to verify the influence of the linker on the biological profile of the compounds. All tested compounds demonstrated significant antiproliferative effects against solid and hematological human tumor cell lines. Among them, 11b exhibited nanomolar potency against hematological tumor cells including Jurkat and Namalwa, with IC50 values of 40 and 200 nM, respectively. Cellular and molecular proliferation studies, in presence of compounds 11a-d, showed significant cell growth arrest, apoptosis induction, and up to 43-fold selective cytotoxicity for leukemia cells versus non-tumorigenic cells. Moreover, compounds 11a-d increased acetylated α-tubulin expression levels, which is phenotypically consistent with HDAC inhibition, and indirectly induced DNA damage. In vitro enzymatic assays performed for 11b revealed a potent HDAC6 inhibitory activity (IC50: 8.1 nM) and 402-fold selectivity over HDAC1. Regarding SAR analysis, the distance between the hydroxamate moiety and the aromatic ring as well as the presence of the double bond in the cinnamyl linker were the most relevant chemical feature for the antiproliferative activity of the series. Molecular modeling studies suggest that cinnamyl hydroxamate is the best moiety of the series for binding HDAC6 catalytic pocket whereas exploration of Ser568 by the urea connecting unity (CU) might be related with the selectivity observed for the cinnamyl derivatives. In summary, cinnamyl hydroxamate derived compounds with HDAC6 inhibitory activity exhibited cell growth arrest and increased apoptosis, as well as selectivity to acute lymphoblastic leukemia cells. This study explores interesting compounds to fight against neoplastic hematological cells.

Novel SIRPα Antibodies That Induce Single-Agent Phagocytosis of Tumor Cells while Preserving T Cells.

The signal regulatory protein α (SIRPα)/CD47 axis has emerged as an important innate immune checkpoint that enables cancer cell escape from macrophage phagocytosis. SIRPα expression is limited to macrophages, dendritic cells, and neutrophils-cells enriched in the tumor microenvironment. In this study, we present novel anti-SIRP Abs, SIRP-1 and SIRP-2, as an approach to targeting the SIRPα/CD47 axis. Both SIRP-1 and SIRP-2 bind human macrophage SIRPα variants 1 and 2, the most common variants in the human population. SIRP-1 and SIRP-2 are differentiated among reported anti-SIRP Abs in that they induce phagocytosis of solid and hematologic tumor cell lines by human monocyte-derived macrophages as single agents. We demonstrate that SIRP-1 and SIRP-2 disrupt SIRPα/CD47 interaction by two distinct mechanisms: SIRP-1 directly blocks SIRPα/CD47 and induces internalization of SIRPα/Ab complexes that reduce macrophage SIRPα surface levels and SIRP-2 acts via disruption of higher-order SIRPα structures on macrophages. Both SIRP-1 and SIRP-2 engage FcγRII, which is required for single-agent phagocytic activity. Although SIRP-1 and SIRP-2 bind SIRPγ with varying affinity, they show no adverse effects on T cell proliferation. Finally, both Abs also enhance phagocytosis when combined with tumor-opsonizing Abs, including a highly differentiated anti-CD47 Ab, AO-176, currently being evaluated in phase 1 clinical trials, NCT03834948 and NCT04445701 SIRP-1 and SIRP-2 are novel, differentiated SIRP Abs that induce in vitro single-agent and combination phagocytosis and show no adverse effects on T cell functionality. These data support their future development, both as single agents and in combination with other anticancer drugs.

Synthesis and in vitro study of antiproliferative benzyloxy dihydropyrimidinones.

In this study, we report on antiproliferative benzyloxy dihydropyrimidinones (DHPMs) produced by the Biginelli reaction of benzyloxy benzaldehyde, urea, and diverse 1,3-diones. The reaction was catalyzed by lanthanum triflate and completed within 1-1.5 h, with 74-97% yield. The antiproliferative assay was carried out for all synthesized dihydropyrimidinones against six human solid tumor cell lines. Six compounds showed good antiproliferative activity with GI50 values below 5 μM. Among all the synthesized compounds, the most potent derivative showed good antiproliferative activity against all cell lines with GI50 values in the range of 1.1-3.1 μM. These DHPMs comply with druglikeness. Furthermore, ADMET prediction and the effect of P-glycoprotein on the antiproliferative activity were also studied. Overall, our method allows eco-friendly access to benzyloxy DHPMs as potential anticancer drugs.

Biological Profiling of Semisynthetic C19-Functionalized Ferruginol and Sugiol Analogues

The abietane-type diterpenoids are significant bioactive compounds exhibiting a varied range of pharmacological properties. In this study, the first synthesis and biological investigation of the new abietane-diterpenoid (+)-4-epi-liquiditerpenoid acid (8a) together with several of its analogs are reported. The compounds were generated from the readily available methyl callitrisate (7), which was obtained from callitrisic acid present in Moroccan Sandarac resin. A biological evaluation was conducted to determine the effects of the different functional groups present in these molecules, providing basic structure–activity relationship (SAR) elements. In particular, the ferruginol and sugiol analogs compounds 10–16 were characterized by the presence of a phenol moiety, higher oxidization states at C-7 (ketone), and the hydroxyl, methyl ester or free carboxylic acid at C19. The biological profiling of these compounds was investigated against a panel of six human solid tumor cell lines (HBL-100, A549, HeLa, T-47D, SW1573 and WiDr), four parasitic Leishmania species (L. donovani, L. infantum, L. guyanensis and L. amazonensis) and two malaria strains (3D7 and K1). Furthermore, the capacity of the compounds to modulate gamma-aminobutyric acid type A (GABAA) receptors (α1β2γ2s) is also described. A comparison of the biological results with those previously reported of the corresponding C18-functionalized analogs was conducted.

Structure-based design of highly selective 2,4,5-trisubstituted pyrimidine CDK9 inhibitors as anti-cancer agents.

Cyclin-dependent kinases (CDKs) are a family of Ser/Thr kinases involved in cell cycle and transcriptional regulation. CDK9 regulates transcriptional elongation and this unique property has made it a potential target for several diseases. Due to the conserved ATP binding site, designing selective CDK9 inhibitors has been challenging. Here we report our continued efforts in the optimization of 2,4,5-tri-substituted pyrimidine compounds as potent and selective CDK9 inhibitors. The most selective compound 30m was >100-fold selective for CDK9 over CDK1 and CDK2. These compounds showed broad anti-proliferative activities in various solid tumour cell lines and patient-derived chronic lymphocytic leukaemia (CLL) cells. Decreased phosphorylation of the carboxyl terminal domain (CTD) of RNAPII at Ser-2 and down-regulation of anti-apoptotic protein Mcl-1 were confirmed in both the ovarian cancer model A2780 and patient-derived CLL cells.

Abstract PO041: Landscape of molecular events regulating tumor cell responses to natural killer cells

Abstract Natural killer (NK) cells exhibit potent activity in pre-clinical models of diverse hematologic malignancies and solid tumors and infusion of high numbers of NK cells, either autologous or allogeneic, after their ex vivo expansion and activation, has been feasible and safe in clinical studies. To systematically define molecular features in human tumor cells which determine their degree of sensitivity to human allogeneic NK cells, we quantified the NK cell responsiveness of hundreds of molecularly-annotated “DNA-barcoded” solid tumor cell lines in multiplexed format (PRISM; Profiling Relative Inhibition Simultaneously in Mixtures approach), correlating cytotoxicity scores for each cell line with the CCLE transcriptional data (RNA-seq), to reveal genes that are associated with resistance or sensitivity to NK cells. In addition, we applied genome-scale CRISPR-based gene editing screens in several solid tumor cell lines to interrogate, at a functional level, which genes regulate tumor cell response to NK cells. Based on these orthogonal studies, NK sensitive tumor cells tend to exhibit high levels of the NK cell-activating ligand B7-H6 (NCR3LG1); low levels of the inhibitory ligand HLA-E; microsatellite instability (MSI) status; high transcriptional signature for chromatin remodeling complexes and low antigen presentation machinery genes. Treatment with an HDAC inhibitor reduced the sensitivity of SW620 colon cancer cells, increased antigen presentation machinery, including HLA-E, and reduced B7-H6. Importantly, we observe that transcriptional signatures of NK cell-sensitive tumor cells correlate with immune checkpoint inhibitor resistance in clinical samples. Strikingly, comprehensive analysis of the CCLE transcriptional signatures revealed that cell lines with mesenchymal-like program tend to be more sensitive to NK cells treatment, compared with cell lines of epithelial-like program. Indeed, mesenchymal tumors tend to have lower expression of antigen presentation machinery in both CCLE and TCGA, suggesting a link between these two machieneries. This study provides a comprehensive map of mechanisms regulating tumor cell responses to NK cells, with implications for future biomarker-driven applications of NK cell immunotherapies. Citation Format: Michal Sheffer, Emily Lowry, Nicky Beelen, Minasri Borah, Suha Naffar-Abu Amara, Chris C. Mader, Jennifer Roth, Aviad Tsherniak, Olga Dashevsky, Sara Gandolfi, Samantha Bender, Jordan Bryan, Cong Zhu, Li Wang, Ricardo De-Matos Simoes, Channing Yu, Yiguo Hu, Olli Dufva, Marios Giannakis, Todd Golub, Rizwan Romee, Satu Mustjoki, Aedin C. Culhane, Lotte Wieten, Constantine S. Mitsiades. Landscape of molecular events regulating tumor cell responses to natural killer cells [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO041.

Studying Histone Deacetylase Inhibition and Apoptosis Induction of Psammaplin A Monomers with Modified Thiol Group.

Psammaplin A (PsA) is a bromotyrosine disulfide dimer with histone deacetylase (HDAC) inhibition and acts through reduced monomer PsA-SH. We studied the connection of HDAC inhibition, cell growth inhibition, and apoptosis induction of PsA-SH by modifying the -SH group with deletion (6a) or replacement with hydroxamic acid (10b) or benzamide (12g). PsA-SH inhibits HDAC1/2/3 and 6a loses the HDAC inhibition ability. 10b inhibits HDAC1/2/3/6 while 12g shows selective inhibition of HDAC3. PsA-SH and 10b, but neither 6a nor 12g, induce apoptosis in human leukemia HL-60 cells associated with increased acetylation of Histone H3. PsA-SH and 10b inhibit growth of several solid tumor cell lines in vitro and Lewis lung cancer cell growth in vivo. PsA-SH is a simple scaffold for developing selective HDAC inhibitors and induces apoptosis through inhibiting HDAC1/2.

Pan-Cancer Transcriptional Models Predicting Chemosensitivity in Human Tumors.

Motivation:Despite increasing understanding of the molecular characteristics of cancer, chemotherapy success rates remain low for many cancer types. Studies have attempted to identify patient and tumor characteristics that predict sensitivity or resistance to different types of conventional chemotherapies, yet a concise model that predicts chemosensitivity based on gene expression profiles across cancer types remains to be formulated. We attempted to generate pan-cancer models predictive of chemosensitivity and chemoresistance. Such models may increase the likelihood of identifying the type of chemotherapy most likely to be effective for a given patient based on the overall gene expression of their tumor.Results:Gene expression and drug sensitivity data from solid tumor cell lines were used to build predictive models for 11 individual chemotherapy drugs. Models were validated using datasets from solid tumors from patients. For all drug models, accuracy ranged from 0.81 to 0.93 when applied to all relevant cancer types in the testing dataset. When considering how well the models predicted chemosensitivity or chemoresistance within individual cancer types in the testing dataset, accuracy was as high as 0.98. Cell line–derived pan-cancer models were able to statistically significantly predict sensitivity in human tumors in some instances; for example, a pan-cancer model predicting sensitivity in patients with bladder cancer treated with cisplatin was able to significantly segregate sensitive and resistant patients based on recurrence-free survival times (P = .048) and in patients with pancreatic cancer treated with gemcitabine (P = .038). These models can predict chemosensitivity and chemoresistance across cancer types with clinically useful levels of accuracy.

A Mechanistic Investigation on the Anticancer Properties of SYA013, a Homopiperazine Analogue of Haloperidol with Activity against Triple Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) is one of the most malignant cancers associated with early metastasis, poor clinical prognosis, and high recurrence rate. TNBC is a distinct subtype of breast cancer that lacks estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 receptors (HER2). Development of effective TNBC therapies has been limited partially due to the lack of specific molecular targets and chemotherapy involving different cytotoxic drugs suffers from significant side effects and drug-resistance development. Therefore, there is an unmet need for the development of novel and efficient therapeutic drugs with reduced side effects to treat TNBC. We have previously reported that certain analogues of haloperidol (a typical antipsychotic drug used for treating mental/mood disorders such as schizophrenia and bipolar disorder) suppress the viability of a variety of solid tumor cell lines, and we have identified 4-(4-(4-chlorophenyl)-1,4-diazepan-1-yl)-1-(4-fluoro-phenyl)butan-1-one (SYA013) with such antiproliferative properties. Interestingly, unlike haloperidol, SYA013 shows moderate selectivity toward σ2 receptors. In this study, we explored the potential of SYA013 in modulating the important biological events associated with cell survival and progression as well as the mechanistic aspects of apoptosis in a representative TNBC cell line (MDA-MB-231). Our results indicate that SYA013 inhibits the proliferation of MDA-MB-231 cells in a concentration-dependent manner and suppresses cell migration and invasion. Apoptotic studies were also conducted in MDA-MB-468 cells (cells derived from a 51-year old Black female with metastatic adenocarcinoma of the breast.). In addition, we have demonstrated that SYA013 induces MDA-MB-231 cell death through the intrinsic apoptotic pathway and may suppress tumor progression and metastasis. Taken together, our study presents a mechanistic pathway of the anticancer properties of SYA013 against TNBC cell lines and suggests a potential for exploring SYA013 as a lead agent for development against TNBC.

Selective Vulnerability to Pyrimidine Starvation in Hematologic Malignancies Revealed by AG-636, a Novel Clinical-Stage Inhibitor of Dihydroorotate Dehydrogenase.

Agents targeting metabolic pathways form the backbone of standard oncology treatments, though a better understanding of differential metabolic dependencies could instruct more rationale-based therapeutic approaches. We performed a chemical biology screen that revealed a strong enrichment in sensitivity to a novel dihydroorotate dehydrogenase (DHODH) inhibitor, AG-636, in cancer cell lines of hematologic versus solid tumor origin. Differential AG-636 activity translated to the in vivo setting, with complete tumor regression observed in a lymphoma model. Dissection of the relationship between uridine availability and response to AG-636 revealed a divergent ability of lymphoma and solid tumor cell lines to survive and grow in the setting of depleted extracellular uridine and DHODH inhibition. Metabolic characterization paired with unbiased functional genomic and proteomic screens pointed to adaptive mechanisms to cope with nucleotide stress as contributing to response to AG-636. These findings support targeting of DHODH in lymphoma and other hematologic malignancies and suggest combination strategies aimed at interfering with DNA-damage response pathways.

An Integrated in silico Approach and in vitro Study for the Discovery of Small-Molecule USP7 Inhibitors as Potential Cancer Therapies.

The ubiquitin-specific protease 7 (USP7) is a highly promising well-validated target for a variety of malignancies. USP7 is critical in regulating the tumor suppressor p53 along with numerous epigenetic modifiers and transcription factors. Previous studies showed that USP7 inhibitors led to increased levels of p53 and anti-proliferative effects in hematological and solid tumor cell lines. Thus, this study aimed to identify potent and safe USP7 hit inhibitors as potential anti-cancer therapeutics via an integrated computational approach that combines pharmacophore modeling, molecular docking, molecular dynamics (MD) simulations and post-MD free energy calculations. In this study, the crystal structure of USP7 has been extensively investigated using a combination of three different chemical pharmacophore modeling approaches. We then screened ∼220.000 drug-like small molecule library and the hit ligands predicted to be nontoxic were evaluated further. The identified hits from each pharmacophore modeling study were further examined by 1-ns short MD simulations and MM/GBSA free energy analysis. In total, we ran 1 ns MD simulations for 1137 selected on small compounds. Based on their average MM/GBSA scores, 18 ligands were selected for 50 ns MD simulations along with one highly potent USP7 inhibitor used as a positive control. The in vitro enzymatic inhibition assay testing of our lead 18 molecules confirmed that 7 of these molecules were successful in USP7 inhibition. Screening results showed that within the used screening approaches, the most successful one was structure-based pharmacophore modeling with the success rate of 75 %. The identification of potent and safe USP7 small molecules as potential inhibitors is a step closer to finding appropriate effective therapies for cancer. Our lead ligands can be used as a scaffold for further structural optimization and development, enabling further research in this promising field.

Discovery of 12O-A Novel Oral Multi-Kinase Inhibitor for the Treatment of Solid Tumor.

A novel series of pyrimidine-benzotriazole derivatives have been synthesized and evaluated for their anticancer activity against human solid tumor cell lines. The most promising molecule 12O was identified for its excellent antiproliferative activities, especially against the SiHa cell line with IC50 value as 0.009 μM. Kinase inhibition assay assessed 12O was a potential multi-kinase inhibitor, which possessed potent inhibitory activities against cyclin-dependent kinases (CDKs) and fms-like tyrosine kinase (FLT) with IC50 values in the nanomolar range. Molecular docking studies illustrated that the introduction of triazole moiety in 12O was critical for CDKs inhibition. In addition, 12O inhibited cancer cell proliferation, colony-formation, and cell cycle progression and provoked apoptotic death in vitro. In an SiHa xenograft mouse model, a once-daily dose of compound 12O at 20 mg/kg significantly suppressed the tumor growth without obvious toxicity. Taken together, 12O provided valuable guide for further structural optimization for CDKs and FLT inhibitors.

Antitumor Effect of Sugar-Modified Cytosine Nucleosides on Growth of Adult T-Cell Leukemia Cells in Mice.

Adult T-cell leukemia (ATL) is a CD4+ T-cell neoplasm caused by human T-cell leukemia virus type I. As the prognosis for patients with ATL remains extremely poor due to resistance to conventional chemotherapy regimens, introduction of novel therapeutic agents is needed. Previous studies have reported that nucleosides 2′-deoxy-2′-methylidenecytidine (DMDC) and its derivative 2′-deoxy-2′-methylidene-5-fluorocytidine (FDMDC) exhibit antitumor activities in T-cell acute lymphoblastic leukemia (T-ALL) and solid tumor cell lines. Another nucleoside, 1-(2-azido-2-deoxy-β-D-arabinofuranosyl)cytosine (cytarazid), is considered a therapeutic drug with antitumor activity in human solid tumors. In this study, we investigated the effects of these nucleosides on cell growth in vitro and in vivo using relevant leukemia cell lines and NOD/Shi-scid, IL-2Rgnull (NOG) mice, respectively. The nucleosides demonstrated significant cytotoxic effects in ATL and T-ALL cell lines. Intraperitoneal administration of FDMDC and DMDC into tumor-bearing NOG mice resulted in significant suppression of tumor growth without lethal side effects. Our findings support a therapeutic application of these nucleosides against tumor progression by targeting DNA polymerase-dependent DNA synthesis in patients with ATL.

Multiplexed Engineered, Off-the-Shelf T Cells Carrying Three Tumor-Associated Antigen-Targeting Modalities: CAR + Pan-Tumor Targeting TCR + CD16 Fc Receptor

Autologous chimeric antigen receptor (CAR)-T cell therapy has shown great promise in various hematologic malignancies. However, the complexities associated with immune cell evasion are prevalent causes of disease relapse in many cancers. With the advent of pluripotent stem cell (iPSC)-derived CAR-T cells, many factors that hamper therapeutic efficacy of CAR-T cells can be addressed through multiplexed engineering at the clonal level. This includes enhanced potency, increased capacity for multi-antigen targeting, and the consistency of a clonally derived engineered cellular product for off-the-shelf patient administration. In particular, strategies to mitigate antigen escape and address tumor heterogeneity may help promote durable responses. To combine the potent targeted therapy of the CAR with universal targeting of secondary and tertiary antigens, we expressed an MR1 clonal T cell receptor (TCR) and a high-affinity, non-cleavable CD16 Fc receptor (hnCD16) in our iPSC-derived CAR19 T cells (CAR19-iT cells) directed to leukemia and lymphoma and CAR-MICA/B T cells directed to solid tumors. The MR1-TCR allows highly specific recognition of tumor associated antigen presented by the MR1 protein. The non-polymorphic MHC class I-related protein MR1 is widely expressed with minimal variability among patients and enables the unique prospect to be a universal cancer immunotherapy by using the cognate MR1-TCR. The hnCD16 Fc receptor has been shown to improve antibody-dependent cellular cytotoxicity (ADCC) leveraging the broad range of available therapeutic monoclonal antibodies to target clinically validated tumor antigens. A preliminary assessment demonstrated that MR1-TCR overexpressed in T cells allowed for enhanced recognition of multiple hematological and solid tumor cell lines. Notably, prominent target specific killing was seen in A549 lung carcinoma cells (>75% reduction in total viable cells) with the directed cytotoxicity specifically inhibited by an MR1 blocking antibody. Next, in vitro functional testing was performed on the engineered CAR19-iT cells in co-culture assays where we measured killing of tumor cells via MR1-TCR engagement and via hnCD16 mediated ADCC. Specifically, we show that CAR19-iT cells expressing hnCD16 can be efficiently directed to lyse CD20+ Raji cells in the presence of rituximab or HER2+ SKOV3 cells in the presence of Herceptin, demonstrating the potential to target both hematological malignancies and solid tumors with one target modality in combination with various monoclonal antibodies. Moreover, CAR19-iT cells expressing either MR1-TCR or hnCD16 show the ability to control growth of CD19 KO lymphoma cells in the co-culture assays, further highlighting the unique ability to elicit multiple ways to target antigen escape. Further in vitro and in vivo combinatorial targeting studies focused on antigen escape and tumor heterogeneity are ongoing and will be discussed. In summary, the advances presented here demonstrate that both the MR1-TCR and hnCD16 modalities synergize with CAR-iT cells as an off-the-shelf therapeutic that can provide durable responses and enable broad applicability for targeting of additional tumor antigens where single-agent therapeutics fail to provide clinical benefit for patients. Disclosures Nguyen: Fate Therapeutics, Inc.: Current Employment. Peralta:Fate Therapeutics, Inc.: Current Employment. Lu:Fate Therapeutics, Inc.: Current Employment. Sung:Fate Therapeutics, Inc.: Current Employment. Lee:Fate Therapeutics, Inc.: Current Employment.

Human Placental CD34+-Derived Natural Killer Cells with High Affinity and Cleavage Resistant CD16 (CYNK-101) for ADCC Mediated Cancer Immunotherapy

CYNK-101, an allogeneic off-the-shelf human placental CD34+-derived natural killer (NK) cell product, is genetically modified to express a variant of CD16 (FcγRIII) via lentiviral vector transduction. When targeting tumors with monoclonal antibodies, NK cells are key effectors of antibody dependent cellular cytotoxicity (ADCC) following recognition of antibody Fc by CD16. CYNK-101 expresses the CD16 variant (CD16VP) which has a high-affinity due to a Valine at amino acid position 158 along with proteolytic cleavage resistance imparted by Proline at amino acid position 1971,2,3,4. We hypothesize that expressing CD16VP enhances anti-tumor activity of CYNK-101 in combination with monoclonal antibody therapy. Reported here are the in vitro and ex vivo results of evaluating CYNK-101 cytotoxicity against human epidermal growth factor receptor 2 (HER2)+ solid tumors in combination with Trastuzumab, an anti-HER2 monoclonal antibody. Human placental CD34+ cells were transduced with lentivirus expressing CD16VP, and cultured in the presence of cytokines, including TPO, SCF, Flt3L, IL-7, IL-15, and IL-2, to generate CYNK-101 cells. Lentiviral transduction with CD16VP achieved high expression efficiency in multiple placental CD34+ donors. These cells (n=7) expanded at 12041 ± 6394-fold and differentiated into CYNK-101 being >90% CD56+CD3-. CYNK-101 expressed 74.1 ± 5.6% (n=7) of CD16 at the end of cultivation. Proteolytic cleavage resistance conferred by CD16VP in CYNK-101 was evaluated by activating NK cells with PMA/ionomycin (PMAi) to induce CD16 shedding. While 4h PMAi treatment resulted in >89% CD16 cleavage on non-transduced NK cells, <11% cleavage was observed on CYNK-101 cells, demonstrating CD16 shedding resistance. The in vitro anti-tumor activity of CYNK-101 against HER2+ solid tumor cell lines was assessed in combination with Trastuzumab. At effector to target (E:T) ratio of 0.6:1, CYNK-101 (n=7 donors) showed enhanced lysis of NCI-N87 gastric cancer cells in the presence of Trastuzumab compared to that of IgG control, 41.2 ± 11.1% vs. 5.3 ± 4.7% at 4h, 91.3 ± 15.0% vs. 7.5 ± 9.3% at 24h, respectively (p<0.005). Increased production of GM-CSF, IFN-γ, and TNF-α was shown in CYNK-101 plus Trastuzumab against NCI-N87 at 24h compared to that of IgG control. Overall, the enhanced ADCC activity of CYNK-101 in combination with Trastuzumab has been demonstrated against both HER2+ gastric cancer cell lines, NCI-N87 and OE-19; and HER2+ breast cancer cell lines, AU565, BT-474, HCC-1954, SKBR-3, and ZR-75-30. To further evaluate ex vivo ADCC activity of CYNK-101, CYNK-101 cells were intravenously (IV) injected at 2x107 into busulfan-pretreated NOD-scid IL2Rγnull immunodeficient (NSG) mice at Day 0. Recombinant human IL-15 was intraperitoneally injected at Days 0, 2, 4, 6, 8, 10 and 12. At Day 13, CYNK-101 cells were isolated from mouse livers (ex vivo-CYNK-101) and assessed for phenotype and ADCC activity. Ex vivo-CYNK-101 exhibited enhanced cytotoxicity against NCI-N87 in combination with Trastuzumab compared to that of IgG control (30.9% vs. 6.6% at 4h, 90.7% vs. 9.1% at 24h) at the E:T ratio of 0.5:1, as well as increased cytokine productions of GM-CSF, IFN-γ, and TNF-α at 24h. Compared to pre-infusion CYNK-101, ex vivo-CYNK-101 showed not only a higher ADCC activity against NCI-N87, but also a more matured NK cell phenotype with increased expression of CD16, KIR, NKG2D, CD94, and CD11a. Besides, these ex vivo-CYNK-101 cells demonstrated CD16 shedding resistance post PMAi stimulation. In summary, our results demonstrated enhanced in vitro and ex vivo ADCC activities of CYNK-101 in combination with Trastuzumab against HER2+ solid tumors. CYNK-101 exhibited high resistance to CD16 shedding in vitro and ex vivo. Further maturation of CYNK-101 cells post IV infusion was accompanied by ADCC activity enhancement. Evaluation of in vivo ADCC activity of CYNK-101 in combination with Trastuzumab will include both subcutaneous and orthotopic gastric cancer models. Disclosures Kang: Celularity Inc.: Current Employment. Raitman:Celularity Inc.: Current Employment. Rotondo:Celularity Inc.: Current Employment. Gleason:Celularity Inc.: Current Employment, Current equity holder in private company. He:Celularity Inc.: Current Employment, Current equity holder in private company. Somanchi:Celularity Inc.: Current Employment, Current equity holder in private company. Rousseva:Celularity Inc.: Current Employment. Guo:Celularity Inc.: Current Employment. Rana:Celularity Inc.: Current Employment. DiFiglia:Celularity Inc.: Current Employment. Ye:Celularity Inc.: Current Employment, Patents & Royalties. Mahlakõiv:Celularity Inc.: Current Employment. Van Der Touw:Celularity Inc.: Current Employment. Hariri:Celularity Inc.: Current Employment, Current equity holder in private company. Zhang:Celularity Inc.: Current Employment, Current equity holder in private company.